Agents and methods for treating pancreatic ductal adenocarcinomas

ABSTRACT

It has been discovered that NAD+-dependent histone deacetylase SIRT6 is critical for suppression of PDAC by con trolling the expression of Lin28b, which is a negative regulator of let-7 microRNA. Specifically, SIRT6 loss results in histone hyperacetylation at the Lin28b promoter, Myc recruitment, and pronounced induction of Lin28b and downstream let-7 target genes, HMGA2, IGF2BP1, and IGF2BP3. This invention relates generally to agents and methods of reducing expression or activity of Lin28b to treat (aggressive) PDAC in a subject.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Application No. 62/141,317,filed on Apr. 1, 2015, which is incorporated by reference in itsentirety.

TECHNICAL FIELD

This invention relates to agents and methods for treating (aggressive)pancreatic ductal adenocarcinoma (PDAC).

BACKGROUND

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethalof all human malignancies and is responsible for hundreds of thousandsof deaths each year. Thus, there is an urgent need to improve ourunderstanding of the molecular underpinnings that drive PDAC initiation,progression and metastasis and to leverage that understanding towardbetter therapeutic options. The current model proposes that a series ofgenetic alterations results in a stepwise progression throughincreasingly dysplastic precursor lesions, or pancreatic intraepithelialneoplasias (PanINs), toward invasive and finally metastatic PDAC.Initiating events identified in early PanIN lesions (PanIN I) includemutations and/or amplification in the KRAS oncogene and the loss ofCDKN2A (p16INK4A) tumor suppressor gene, present in >90% and >50% ofPDAC/PanINs, respectively (Ryan et al., 2014). Higher grade lesions(PanIN III) and invasive PDAC may accumulate additional genetic lesions,including inactivation of TP53 and TGFβ pathway components (SMAD4,TGFβR1, and TGFβR2), found in 60-70% and 50% of PDAC, respectively (Ryanet al., 2014). However, this fundamental model of PDAC pathogenesis,which is recapitulated in genetically engineered mice, has failed toidentify either critical pathways that may be effectively targeted inthe clinic or relevant molecular subsets for improved prognosis andstratification of patients toward a more effective therapy.

In addition to the above well-characterized genetic aberrations, it isbecoming increasingly apparent that the dysregulation of epigeneticmodifiers is central to the initiation and progression of human PDAC aswell as many other tumors. Genomic deletions, mutations, andrearrangements recurrently targeting genes encoding components of theSWitch/Sucrose NonFermentable (SWI/SNF) chromatin remodeling complex,including all three putative DNA binding subunits (ARID1A, ARID1B, andPBRM1) and both enzymatic subunits (SMARCA2 and SMARC4), have beenrecently identified in at least 10-15% of PDAC cases. Additionally,mutations in the histone methyltransferase mixed-lineage leukemiaprotein 2 & 3 (MLL2 & MLL3) and the histone demethylase Kdm6a have beenidentified in 5-10% of PDAC (Ryan et al., 2014). However, since thesechromatin-modifying enzymes may simultaneously regulate thetranscription of thousands of genes by altering chromatin structurethroughout the genome or may be involved in other cellular functionssuch as DNA repair and replication, the mechanisms by which theseproteins control tumorigenesis have been difficult to elucidate.Specifically, whether these enzymes regulate an individual target geneor set of genes to drive survival, proliferation, cellulartransformation, metabolic adaptations or invasive functions in PDAC isunknown; yet this understanding is critical to our ability to leveragedata from the molecular profiling of human tumors to identify newtherapeutic opportunities in molecularly-defined subsets of disease.

SUMMARY

This invention is based, at least in part, on the discovery that Sirtuin6 (SIRT6) acts a potent tumor suppressor in genetically-engineered mousemodels (GEMMS) of oncogenic Kras-driven PDAC, regardless of p53 status.Surprisingly, loss of SIRT6 did not accelerate PDAC tumorigenesis byenhancing aerobic glycolysis, as observed in colon cancer. Instead, lossof SIRT6 results in reactivation of the oncofetal protein Lin28b in bothhuman and murine PDAC. Importantly, this de-repression results in theupregulation of numerous let-7 target genes and is critical for thesurvival of SIRT6-deficient PDAC. The SIRT6/Lin28b axis is a novelpathway in PDAC carcinogenesis and a molecularly defined subset that maybenefit from therapeutic intervention.

In one aspect, the present disclosure features use of a Lin28b inhibitorfor treating PDAC in a subject. In one aspect, the present disclosureprovides methods of treating PDAC in a subject by administering atherapeutically effective amount of a Lin28b inhibitor to the subject.

In another aspect, the present disclosure features methods of diagnosingand optionally treating PDAC in a subject, wherein the methods includeproviding a sample comprising pancreatic cells from the subject;performing an assay to determine a level of Lin28b and/or SIRT6expression in the sample; comparing the level of Lin28b and/or SIRT6expression in the sample to a reference level of Lin28b and/or SIRT6expression, respectively; identifying a subject who has a level ofLin28b expression in the sample above the reference level as having PDACor having an increased risk of developing PDAC; identifying a subjectwho has a level of SIRT6 expression in the sample below the referencelevel as having PDAC or having an increased risk of developing PDAC; andoptionally administering a treatment for PDAC to the identified subjectwho has a level of Lin28b expression in the sample that is above thereference level and/or to the identified subject who has a level ofSIRT6 expression in the sample that is below the reference level,wherein the treatment comprises an inhibitory nucleic acid effective tospecifically reduce expression of Lin28b. In some embodiment, the levelof Lin28b and/or SIRT6 expression in the sample is determined byquantitative PCR, flow cytometry, or quantitative immunoassay.

The uses and methods are effective for a variety of subjects includingmammals, e.g., humans and other animals, such as laboratory animals,e.g., mice, rats, rabbits, or monkeys, or domesticated and farm animals,e.g., cats, dogs, goats, sheep, pigs, cows, or horses.

In some embodiments, the Lin28b inhibitor can be an inhibitory nucleicacid effective to specifically reduce expression of Lin28b, e.g., asmall interfering RNA molecule, antisense nucleic acid, locked nucleicacid (LNA) molecule, peptide nucleic acid (PNA) molecule, or ribozyme.

In some embodiments, the inhibitory nucleic acid is 5 to 40 bases inlength (optionally 12-30, 12-28, or 12-25 bases in length). In oneembodiment, the inhibitory nucleic acid has a sequence of5′-GCCTTGAGTCAATACGGGTAA-3′ (SEQ ID NO:3).

In some embodiments, the inhibitory nucleic acid is 10 to 50 bases inlength.

In some embodiments, the inhibitory nucleic acid comprises a basesequence at least 90% complementary to at least 10 bases of the RNAsequence.

In some embodiments, the inhibitory nucleic acid comprises a sequence ofbases at least 80% or 90% complementary to, e.g., at least 5-30, 10-30,15-30, 20-30, 25-30 or 5-40, 10-40, 15-40, 20-40, 25-40, or 30-40 basesof the RNA sequence.

In some embodiments, the inhibitory nucleic acid comprises a sequence ofbases with up to 3 mismatches (e.g., up to 1, or up to 2 mismatches) incomplementary base pairing over 10, 15, 20, 25 or 30 bases of the RNAsequence.

In some embodiments, the inhibitory nucleic acid comprises a sequence ofbases at least 80% complementary to at least 10 bases of the RNAsequence.

In some embodiments, the inhibitory nucleic acid comprises a sequence ofbases with up to 3 mismatches over 15 bases of the RNA sequence.

In some embodiments, the inhibitory nucleic acid is single stranded.

In some embodiments, the inhibitory nucleic acid is double stranded.

In some embodiments, the inhibitory nucleic acid comprises one or moremodifications comprising: a modified sugar moiety, a modifiedinternucleoside linkage, a modified nucleotide and/or combinationsthereof.

In some embodiments, the inhibitory nucleic acid is an antisenseoligonucleotide, LNA molecule, PNA molecule, ribozyme or siRNA.

In some embodiments, the inhibitory nucleic acid is double stranded andcomprises an overhang (optionally 2-6 bases in length) at one or bothtermini.

In some embodiments, the inhibitory nucleic acid is selected from thegroup consisting of antisense oligonucleotides, ribozymes, externalguide sequence (EGS) oligonucleotides, siRNA compounds, micro RNAs(miRNAs); small, temporal RNAs (stRNA), and single- or double-strandedRNA interference (RNAi) compounds.

In some embodiments, the RNAi compound is selected from the groupconsisting of short interfering RNA (siRNA); or a short, hairpin RNA(shRNA); small RNA-induced gene activation (RNAa); and small activatingRNAs (saRNAs).

In some embodiments, the antisense oligonucleotide is selected from thegroup consisting of antisense RNAs, antisense DNAs, and chimericantisense oligonucleotides.

In some embodiments, the modified internucleoside linkage comprises atleast one of: alkylphosphonate, phosphorothioate, phosphorodithioate,alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphatetriester, acetamidate, carboxymethyl ester, or combinations thereof.

In some embodiments, the modified sugar moiety comprises a2′-O-methoxyethyl modified sugar moiety, a 2′-methoxy modified sugarmoiety, a 2′-O-alkyl modified sugar moiety, or a bicyclic sugar moiety.In some embodiments, the inhibitory nucleic acids include 2′-OMe, 2′-F,LNA, PNA, FANA, ENA or morpholino modifications.

The invention provides several advantages. The prophylactic andtherapeutic methods described herein using a Lin28b inhibitor areeffective in treating PDAC and have minimal, if any, side effects.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting. Other features, objects, and advantages of the inventionwill be apparent from the detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. 1A-1H are a panel of eight figures showing loss of SIRT6cooperates with oncogenic Kras to accelerate PDAC. 1A,Immunohistochemistry of SIRT6 in human PDAC samples (left & center)compared to normal pancreas (right) and quantification of IHC scoring(bottom right). 1B, Kaplan-Meier analysis of the indicated PDAC patientsamples based on SIRT6 IHC score (n=120). 1C, Kaplan-Meier analysis ofthe indicated GEMMS showing time until signs of illness necessitatedeuthanasia. All animals euthanized had pancreatic tumors. 1D, Necropsyof Sirt6 Kras^(G12D); p53^(f/); p48-Cre (SIRT6 KO) GEMM euthanized at 13weeks. Top left, Image of abdominal contents showing pancreatic mass andsplenomegaly. Top middle, extracted SIRT6 KO tumor. Upper right,haematoxylin and eosin (H&E) staining showing PDAC histology. Bottomleft, Gross image of liver with metastases. Bottom middle, H&E stain ofliver metastasis. Bottom right, H&E stain of lung metastasis. 1E,Quantification of the number of metastases in the livers or the lungs ofSirt6^(f/f); Kras^(G12D); p53^(f/+); p48-Cre and Sirt6^(+/+);Kras^(G12D); p53^(f/+); p48-Cre GEMMS from the Kaplan-Meier analysis inFIG. 1C. 1F, Kaplan-Meier analysis of the indicated GEMMS showing timeuntil signs of illness necessitated euthanasia. All animals euthanizedhad pancreatic tumors. 1G, Necropsy of Sirt6^(f/f); Kras^(G12D);p53^(+/+) p48-Cre GEMM euthanized at 55 weeks. Top left, Image ofabdominal contents showing pancreatic mass and splenomegaly. Top middle,extracted Sirt6^(f/f); Kras^(G12D); p53^(−/+), p48-Cre pancreatic tumorwith spleen attached. Upper right, haematoxylin and eosin (H&E) stainingshowing PDAC histology. Bottom left, H&E of liver metastasis. Bottomright, H&E stain of lung metastasis. 1H, Quantification of the number ofmetastases in the livers or the lungs of Sirt6^(f/f); Kras^(G12D);p53^(+/+); p48-Cre and Sirt6^(+/+); Kras^(G12D); p53^(+/+); p48-CreGEMMS from the Kaplan-Meier analysis in FIG. 1F. Scale bars, black 50μm, blue 20 μm. *p<0.05; **p<0.01; ***p<0.001.

FIGS. 2A-2P are a series of 16 figures showing that SIRT6 suppressesproliferation of established PDAC through histone deacetylation. 2A,Murine PDAC cells were grown under restrictive, nonadherent conditionsto induce tumor sphere formation, photomicrographs (left) and quantified(right). Two independent cell lines are represented. 2B-2E, Murine PDACcells were engineered to express empty vector, SIRT6 WT or SIRT6 HYcatalytically inactive mutant. 2B, Immunoblot of chromatin extract andWCL. 2C, Growth curve of a representative SIRT6 KO PDAC line. 2D,Quantification of tumor spheres formed by two independent SIRT6 KO PDAClines and grown as in 2A. 2E, Tumor weights (left) and gross image ofSIRT6 KO PDAC cell line grown for 3 weeks as a subcutaneous xenograft(n=5 per genotype). 2F, Immunoblot of WCL in human PDAC cell lines(below) Image J quantifications of SIRT6/actin ratio. 2G-2J, Panc3.27and Panc-1 cells were engineered to express empty vector (vector), SIRT6WT (S6WT), or SIRT6 HY (S6HY) catalytically inactive mutant under adoxycycline (Dox)-inducible system. 2G, Immunoblot of chromatin extract.2H, Immunoblot of chromatin extract from Panc3.27 cells treated with doxfor indicated times. The partial effect of S6HY on H3K56Ac levels after4 days of overexpression likely relates to its partial catalyticactivity. 2I, Proliferation was quantified by trypan blue exclusionassay. 2J, Photomicrographs (left) and quantification of Panc3.27 cellsgrown as in 2A. 2K-2M, HPDE cells were engineered to express emptyvector (shCtl) and shSIRT6 under a doxycycline (Dox)-inducible system.2K, (left) Immunoblot of whole cell lysate. 2L, Quantitative polymerasechain reaction with reverse transcription (qRTPCR) analysis ofglycolytic genes. 2M, FDG-Glucose uptake in HPDE cells. 2N-2P, Panc3.27and Panc-1 cells were engineered to express empty vector (vector), SIRT6WT (S6WT), or SIRT6 HY (S6HY) catalytically inactive mutant under adoxycycline (Dox)-inducible system. 2N, Immunoblot of whole cell lysate.2O, qRTPCR analysis of glycolytic genes. 2P, FDG-Glucose uptake inPanc3.27 cells after cells were treated with dox for the indicatedtimes. *p<0.05; **p<0.01; ***p<0.001.

FIGS. 3A-3J are a series of 10 figures showing that SIRT6 suppressesexpression of the oncofetal protein Lin28b in human and murine PDAC. 3A,Venn diagram of gene promoters decorated by H3K56Ac in SIRT6 WT, SIRT6KO, and SIRT6 KO PDAC cells engineered to express SIRT6 WT as determinedby Chromatin immunoprecipitation (ChIP) sequencing (Seq). 3B,Integrative genomics viewer track of H3K56Ac levels along Lin28bpromoter of the indicated murine PDAC cell lines. 3C, Expression ofLin28b in four independent SIRT6 WT and SIRT6 KO murine PDAC cell linesas measured by qRTPCR; data are presented as mean±s.e.m. between threeindependent experiments. 3D, Immunoblot of chromatin and whole celllysates from individual SIRT6 WT and SIRT6 KO PDAC cell lines. 3E, 3FqRTPCR analysis for expression of LIN28B and SIRT6 in human PDAC celllines displayed as a bar graph; data represent mean±s.e.m. between threeindependent experiments. (3E) and scatter plot (3F) demonstrating aninverse correlation. 3G, Immunohistochemistry of LIN28B and SIRT6 inhuman PDAC samples (left) compared to normal pancreas (right). 3H, 3I,LIN28B levels in Panc3.27 expressing empty vector, S6WT or the S6HYcatalytically inactive mutant as measured by qRTPCR (3H) and immunoblot(3I). 3J, Lin28b levels in two independent SIRT6 KO murine PDAC cellslines expressing empty vector, S6WT or S6HY. Scale bars, black 50 μm,blue 20 μm. *p≦0.05; **p≦0.01; ***p≦0.001.

FIGS. 4A-4J are a series of 10 figures showing that SIRT6 co-repressesMyc-driven transcription of Lin28b through histone deacetylation. 4A,Schematic representation of the human genomic region near thetranscription start site of LIN28B. Putative Myc binding sites areindicated (CACGTG or CATGTG); both sites are conserved between human andmouse. 4B, 4C, ChIP of H3K56Ac (4B) and H3K9Ac (4C) marks followed byamplification of the regions surrounding the Myc binding sites in theLIN28B promoter. 4D, 4F, & 4I, Analysis of three independent SIRT6 KOmurine PDAC cell lines expressing the either shMyc or control hairpinsfor expression of Myc (left) and Lin28b (right) by qRTPCR (4D), cellproliferation (4F), and tumor sphere forming ability (4I). 4E, 4G, 4H, &4J, Analysis of three independent SIRT6^(low) PDAC cell lines expressingthe either shMyc or control hairpins for expression of MYC (left) andLIN28B (right) by qRTPCR (4E), immunoblot of MYC knockdown (4G), cellproliferation (4H), and tumor sphere forming ability (4J). For 4E, 4H, &4J, data are represented as mean±std between triplicates. *p≦0.05;**p≦0.01; ***p≦0.001.

FIGS. 5A-5H are a panel of eight figures showing SIRT6^(low) human PDACcells are addicted to Lin28b. 5A-5H, Human PDAC cell lines with eitherhigh or low levels of SIRT6 expression were treated with shLIN28B versusa control hairpin. 5A, Immunoblot of whole cell lysate for SIRT6 andLIN28B. 5B, Number (left) and size (right) of tumor spheres grown undernonadherent conditions. 5C, Photo-micrographs of tumor spheres. 5D, 5F,Growth curve of SIRT6^(low) (5D) and SIRT6^(high) (5F) human PDAC cells,quantified by MTT assay. 5E, 5G, show visualization of day 6 results inSIRT6^(low) (5E) and SIRT6^(high) (G) human PDAC cells. 5H, Tumorweights of SIRT6^(low) and SIRT6^(high) PDAC lines grown as subcutaneousxenografts (n=5). *p≦0.05; **p≦0.01; ***p≦0.001.

FIGS. 6A-6H are a panel of eight figures showing Lin28b decreases let-7levels and increases Igf2bp1/3 and Hmga2 levels in PDAC. 6A, Immunoblotof whole cell lysate and chromatin of human PDAC cell lines. SIRT6 andACTIN have been reproduced from FIG. 2F for comparison. 6B-6E,SIRT6^(low) and SIRT6^(high) human PDAC cells treated with eithershHMGA2 or control hairpin. Confirmation of HMGA2 knockdown by qRTPCR(6B), immunoblot (6C) growth curves of SIRT6^(low) (Panc3.27, BxPc3 andSu86.86) and SIRT6^(high) (COLO357) human PDAC cell lines (6D).Quantification of sphere diameter of SIRT6^(low) (Panc3.27 and BxPc3)and SIRT6^(high) (COLO357) human PDAC cell lines (left) andrepresentative photo pictomicrographs (right) (6E). 6F-6H, SIRT6^(low)and SIRT6^(high) human PDAC cells treated with either shIGF2BP3 orcontrol hairpins. Confirmation of IGF2BP3 knockdown by qRTPCR (6F),growth curves of SIRT6^(low) (Panc3.27, BxPc3 and Su86.86) andSIRT6^(high) (SUIT2) human PDAC cell lines (6G). Quantification ofsphere diameter and number of SIRT6^(low) (Panc3.27 and BxPc3) andSIRT6^(high) (COLO357) human PDAC cell lines (left) and representativephoto pictomicrographs (right) (6H). For 6B & 6D-6H, data arerepresented as mean±std between triplicates. *p≦0.05; **p≦0.01;***p≦0.001.

FIGS. 7A-7G are a panel of seven figures showing that increasedexpression of LIN28B and let-7 target genes correlates with poorsurvival in PDAC. 7A, Kaplan-Meier analysis of the indicated PDACpatient samples based on LIN28B IHC score (n=120). 7B-7C, Gene setEnrichment Analysis (GSEA) plots showing that human PDAC tumors (7B) andPDAC cell lines from the Cancer Cell line Encyclopedia (CCLE) (7C) withhigh levels of LIN28B (LIN28B^(high)) overexpress many of the genes thatare regulated by let-7. 7D, GSEA plots showing that human LIN28B^(high)PDAC tumors overexpress targets of let-7 which are oncofetal genes. 7E,Correlation of HMGA2 and IGF2BP3 RNA expression in human PDAC samplesfrom the TCGA pancreatic cancer dataset. 7F, Kaplan-Meier survivalcurves for HMGA2 (left) and IGF2BP3 (right) in human pancreatic cancerdatasets from the TCGA. 7G, Model for SIRT6 loss in PDAC pathogenesis.*p≦0.05; **p≦0.01; ***p≦0.001.

DETAILED DESCRIPTION

PDAC is the most common malignancy of the pancreas. PDAC is anaggressive and difficult malignancy to treat. Complete surgical removalof the tumor remains the only chance for cure, however 80-90% ofpatients have disease that is surgically incurable at the time ofclinical presentation. Despite advancing knowledge of the tumor biologyof PDAC, improvement in diagnosis and management, and the rise ofcenters specialized in the care of patients with PDAC, the prognosisremains strikingly poor.

Alterations in epigenetic control are an important hallmark of cancer.Such alterations are thought to endow cells with the plasticity tooverride normal differentiation and growth control programs. Due totheir poor vascularity and dense stroma, PDAC cells must acquiremultiple metabolic adaptations to grow in a hypoperfusedmicroenvironment. SIRT6 is an nicotinamide adenine dinucleotide(NAD)⁺-dependent histone deacetylase which removes acetyl groups fromhistone 3 lysine 9 (H3K9) and histone 3 lysine 56 (H3K56) motifs and haspleiotropic functions including glucose homeostasis, maintenance ofgenome stability, and suppression of cellular transformation(Mostoslaysky et al., 2006; Sebastian et al., 2012; Zhong et al., 2010).These functions are exemplified in both Sirt6-deficient mice, whichexhibit complete loss of subcutaneous fat and lethal hypoglycemia, aswell as SIRT6-deficient cells, which show increased glucose uptake,enhanced glycolysis, anchorage independent growth and tumor formation inan in vivo model of colon cancer (Mostoslaysky et al., 2006; Sebastianet al., 2012). SIRT6 is downregulated in PDAC relative to normal tissueand loss of SIRT6 leads to dysregulation of the PDAC epigenome to driveits growth. By developing novel GEMMS, the present disclosuredemonstrates that ablation of SIRT6 potently cooperates with activatedKras (which is mutated in >90% of human PDAC) to accelerate PDAC onsetand promote metastasis. Mechanistically, loss of SIRT6 results inhyperacetylation of H3K9 and H3K56 at the promoter of the LIN28B gene,creating a more permissive chromatin state and allowing for the Myctranscription factor to drive its expression. This aberrant Lin28bexpression is required for the growth of SIRT6-deficient tumor cells,thus identifying Lin28b as a novel oncogenic driver in this distinctsubset, representing ˜30-40% of human PDACs.

The Lin28/let-7 axis is now recognized as central to maintaining propercell fate and coordinating proliferation, growth, and energy utilizationat the cellular level as well as growth, developmental timing, tissuehomeostasis and metabolism in whole organisms (Thornton and Gregory,2012). While Lin28b is silenced during embryonic development (Moss andTang, 2003; Rybak et al., 2008; Yang and Moss, 2003), it may beaberrantly reactivated in a variety of human cancers (Iliopoulos et al.,2009; Thornton and Gregory, 2012; Viswanathan et al., 2009) bymechanisms that remain poorly understood. Eight loss-of-functiontumor-associated SIRT6 point mutations were recently identified, severalof which specifically abrogated SIRT6 deacetylase activity, and manyhuman cancer cell lines demonstrate copy number loss of the SIRT6 locus(Kugel et al., 2015).

Given the critical roles for Lin28b in stem cell pluripotency, one canspeculate that overexpression of oncofetal proteins reactivate programsof embryonic growth to promote a more “undifferentiated” and therebyaggressive form of pancreatic cancer. Consistently, upregulated genesdownstream of Lin28b, includes the oncofetal RNA-binding proteinsIgf2bp1 & 3 that have been associated with poorly differentiated PDAC.Expression of Igf2bps increase progressively with PDAC tumor stage(Yantiss et al., 2005) and high levels of Igf2bps in PDAC correlate withincreased metastasis and extremely poor survival outcome (Schaeffer etal., 2010; Taniuchi et al., 2014). In this context, signs of acceleratedinitiation (increased number of PanIN) as well as increased metastaticpotential were observed in mice expressing high levels of Lin28b andIgf2bps. Igf2bps also has functions in binding and stabilizing IGF2 andMyc transcripts, thus increasing their translation (Bell et al., 2013;Nielsen et al., 2004; Noubissi et al., 2006). Reinforcing Myc signalingand increasing IGF2 signaling could both serve to encourageproliferation and survival of PDAC cells. Strikingly, knockdown ofIgf2bp3 in multiple independent SIRT6^(low) and SIRT6 KO cell lines wassufficient to significantly inhibit their growth, while having no effecton the growth of SIRT6^(high) and SIRT6 WT lines. Similarly, elevatedprotein expression of HMGA2 in PDAC has been associated with a moreadvanced tumor grade, epithelial to mesenchymal transition, and lymphnode metastases, and this protein also promoted the growth ofSIRT6^(low) but not SIRT6^(high) PDAC cells. Thus, Lin28b appears todrive the growth of SIRT6-deficient PDAC through the inhibition ofmultiple let-7 isoforms, resulting in a coordinated upregulation of alarge number of Lin28b/let-7 target genes, including oncofetal proteinslike IGF2BPs and HMGA2 (FIG. 7G).

There is evidence that reactivation of Lin28b may be the result of amore general mechanism that follows loss of epigenetic barriers. Whenhuman embryonic stem cells were used to model pediatric gliomas withH3.3K27M histone mutations, the gene that was reactivated to the highestextent in response to global H3K27 hypomethylation was LIN28B (Funato etal., 2014). Additionally, prolonged inhibition of the methyltransferaseEZH2, in glioblastoma leads to upregulation of Lin28b expression (deVries et al., 2015). EZH2 acts mainly through trimethylation of histoneH3 lysine27, which is associated with transcriptional repression, thusloss of H3K27 trimethylation in two different contexts lead toupregulation of Lin28b expression. The activity of SIRT6 may provide apreviously unrecognized epigenetic barrier, suppressing the expressionof Lin28b specifically in PDAC. The H3K9 and H3K56 hyperacetylation ofthe Lin28b gene in response to SIRT6 loss may function to inhibit thereciprocal methylation of this histone residue, preventingH3K9Me3-mediated gene silencing, thereby licensing the aberrantre-expression of Lin28b to drive this fatal disease.

Novel therapeutic strategies for Kras-driven cancers such as PDAC havebeen limited by a failure to identify pathways that are specificallyrequired in cancer cells but dispensable in normal tissues. Oncofetalproteins represent attractive targets for such strategies, as they arehighly expressed in embryonic tissues but silenced in normal adultcells. Thus, the findings presented in the present disclosure highlightLin28b as a novel oncogene in PDAC and identify a clinically-relevantand molecularly-defined subset of PDAC, which will benefit from noveltherapeutic approaches aimed at targeting components of the Lin28b/let-7pathway and provide new insights into the epigenetic mechanismsgoverning the reactivation of these developmental programs in cancer.

SIRT6 is a member of a highly conserved family of NAD⁺-dependentdeacetylases with various roles in metabolism, stress resistance, andlife span. Seven examples of SIRT6 are highlighted below in Table 1.SIRT6-deficient mice develop normally but succumb to lethal hypoglycemiaearly in life. The present disclosure relates to the role of SIRT6 as ahistone deacetylase to control the expression of LIN28b. Specifically,SIRT6 is critical for suppression of PDAC. SIRT6 inactivationaccelerates PDAC progression and metastasis via upregulation of Lin28b,a negative regulator of the let-7 microRNA. SIRT6 loss results inhistone hyperacetylation at the Lin28b promoter, Myc recruitment, andpronounced induction of Lin28b and downstream let-7 target genes, HMGA2,IGF2BP1, and IGF2BP3. This epigenetic program defines a distinct subsetrepresenting 30-40% of human PDAC, characterized by poor prognosis andan exquisite dependence on Lin28b for tumor growth. Thus, SIRT6 is animportant PDAC tumor suppressor, and the Lin28b pathway acts as atherapeutic target in a molecularly-defined PDAC subset. Provided hereinare methods for treating PDAC in a subject by administering to thesubject a therapeutically effective amount of a Lin28b inhibitor, e.g.,an inhibitory nucleic acid, e.g., a small interfering RNA molecule,antisense nucleic acid, LNA molecule, PNA molecule, and/or ribozyme.

TABLE 1 SIRT6 orthologs from seven different species along with theirGenBank RefSeq Accession Numbers. Species Nucleic Acid Amino Acid GeneIDHomo sapiens NM_016539.1 NP_057623.1 51548 Mus musculus NM_181586.3NP_853617.1 50721 Rattus norvegicus NM_001031649.1 NP_001026819.1 299638Macaca mulatta NC_007876.1 NW_001106369.1 714545 Pan troglodytesNC_006486.2 NW_001228145.1 737026 Canis lupus NC_006602.2 NW_876272.1485045 familiaris Bos taurus NM_001098084.1 NP_001091553.1 535416

SIRT6 is a nuclear, chromatin-bound protein (Mostoslaysky et al., Cell124:315-329, 2006). Among the sirtuins, SIRT6 deficiency causes the moststriking phenotype. SIRT6 deficient mice are born normally, but ataround 3 weeks of age they develop several acute degenerative processes,dying before one month of age. The defects include a severe metabolicimbalance, with low levels of serum IGF-1, complete loss of subcutaneousfat, lymphopenia, osteopenia, and acute onset of hypoglycemia, leadingto death (Mostoslaysky et al., Cell 124:315-329, 2006). Furthermore,SIRT6 promotes resistance to DNA damage and oxidative stress, andsuppresses genomic instability in mouse cells, in association with arole in base excision DNA repair (BER) (Mostoslaysky et al., Cell124:315-329, 2006). Recent studies have demonstrated that SIRT6 islocated at the telomeres in human cells, and knock-down of SIRT6 inthose cells altered the telomere structure, causing acceleratedsenescence and telomere-dependent genomic instability.

Lin28b is an RNA-binding protein that regulates cell growth anddifferentiation (Lei et al., 2012). Developmental timing inCaenorhabditis elegans is regulated by a heterochronic gene pathway. Theheterochronic gene LIN28 is a key regulator early in the pathway. LIN28encodes an approximately 25-kDa protein with two RNA-binding motifs: aso-called “cold shock domain” (CSD) and a pair of retroviral-type CCHCzinc fingers; it is the only known animal protein with this motifpairing. The CSD is a β-barrel structure that binds single-strandednucleic acids. LIN28 inhibits the biogenesis of a group of microRNAs(miRNAs), among which are the let-7 family miRNAs shown to participatein regulation of the expression of genes involved in cell growth anddifferentiation. LIN28 binds to the terminal loop region ofpri/pre-let-7 and blocks their processing. miRNAs are small RNAmolecules (21-23 nucleotides) that act as negative regulators of geneexpression either by blocking mRNA translation into protein or throughRNA interference. Seven examples of Lin28b are presented below in Table2.

TABLE 2 Lin28b orthologs from seven different species along with theirGenBank RefSeq Accession Numbers. Species Nucleic Acid Amino Acid GeneIDHomo sapiens NM_001004317.3 NP_001004317.1 389421 Mus musculusNM_001031772.2 NP_001026942.1 380669 Gallus gallus NM_001034818.1NP_001029990.1 421786 Rattus norvegicus XM_008773029.1 XP_008771251.1689054 Macaca mulatta XM_015137022.1 XP_014992508.1 696130 Pantroglodytes XM_009451721.1 XP_009449996.1 737588 Canis lupus familiarisXM_539064.5 XP_539064.2 481943

As used herein, “substantially identical” refers to a nucleotidesequence that contains a sufficient or minimum number of identical orequivalent nucleotides to the sequence of SIRT6, such that homologousrecombination can occur. For example, nucleotide sequences that are atleast about 75% identical to the sequence of SIRT6 are defined herein assubstantially identical. In some embodiments, the nucleotide sequencesare about 80%, 85%, 90%, 95%, 99%, or 100% identical.

To determine the percent identity of two sequences, the sequences arealigned for optimal comparison purposes (gaps are introduced in one orboth of a first and a second amino acid or nucleic acid sequence asrequired for optimal alignment, and non-homologous sequences can bedisregarded for comparison purposes). The length of a reference sequencealigned for comparison purposes is at least 80% (in some embodiments,about 85%, 90%, 95%, or 100% of the length of the reference sequence) isaligned. The nucleotides or residues at corresponding positions are thencompared.

When a position in the first sequence is occupied by the same nucleotideor residue as the corresponding position in the second sequence, thenthe molecules are identical at that position. The percent identitybetween the two sequences is a function of the number of identicalpositions shared by the sequences, taking into account the number ofgaps, and the length of each gap, which need to be introduced foroptimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. For example, the percent identity between two amino acidsequences can be determined using the Needleman and Wunsch ((1970) J.Mol. Biol. 48:444-453) algorithm which has been incorporated into theGAP program in the GCG software package, using a Blossum 62 scoringmatrix with a gap penalty of 12, a gap extend penalty of 4, and aframeshift gap penalty of 5.

Methods of Diagnosing or Predicting Risk Based on SIRT6 and/or Lin28bExpression

Included herein are methods for diagnosing and predicting risk ofdeveloping PDAC, or providing a prognosis for a subject who has PDAC.The methods include obtaining a sample comprising (e.g., enriched in)pancreatic cells from a subject, and evaluating the presence and/orlevel of SIRT6 and Lin28b in the sample, and comparing the presenceand/or level with one or more reference levels. The presence and/orlevel of SIRT6 and Lin28b can be evaluated using methods known in theart, e.g., using quantitative immunoassay methods, ELISA, enzymaticassays, flow cytometry with or without cell permeabilization,spectrophotometry, colorimetry, fluorometry, bacterial assays, liquidchromatography, gas chromatography, mass spectrometry, gaschromatography-mass spectrometry (GC-MS), liquid chromatography-massspectrometry (LC-MS), LC-MS/MS, tandem MS, high pressure liquidchromatography (HPLC), HPLC-MS, and nuclear magnetic resonancespectroscopy, or other known techniques for determining the presenceand/or quantity of a protein. The presence and/or level of a nucleicacid can be evaluated using methods known in the art, e.g., usingquantitative PCR methods. In some embodiments, high throughput methods,e.g., protein or gene chips as are known in the art (see, e.g., Ch. 12,Genomics, in Griffiths et al., Eds. Modern genetic Analysis, 1999,W. H.Freeman and Company; Ekins and Chu, Trends in Biotechnology, 1999,17:217-218; MacBeath and Schreiber, Science 2000, 289(5485): 1760-1763;Simpson, Proteins and Proteomics: A Laboratory Manual, Cold SpringHarbor Laboratory Press; 2002; Hardiman, Microarrays Methods andApplications: Nuts & Bolts, DNA Press, 2003), can be used to detect thepresence and/or level of Lin28b in a sample.

In some embodiments, the presence and/or level of SIRT6 and Lin28b iscomparable to the presence and/or level of the protein in the diseasereference, and the subject has one or more symptoms associated withPDAC, e.g., with aggressive PDAC, then the subject is diagnosed with(aggressive) PDAC. Skilled practitioners will recognize that aggressivePDACs can exhibit early onset of liver metastases and/or rapid generaldeterioration of the patient (i.e., cachexia). PDACs in general are veryaggressive tumors, with most PDAC patients (˜90%) dying within one yearof diagnosis. In some embodiments, the subject has no overt signs orsymptoms of PDAC, but the presence and/or level of SIRT6 and/or Lin28bevaluated is comparable to the presence and/or level of SIRT6 and Lin28bin the disease reference, then the subject has an increased risk ofdeveloping (aggressive) PDAC.

Suitable reference values can be determined using methods known in theart, e.g., using standard clinical trial methodology and statisticalanalysis. The reference values can have any relevant form. In somecases, the reference comprises a predetermined value for a meaningfullevel of SIRT6 and Lin28b, e.g., a control reference level thatrepresents a normal level of SIRT6 and Lin28b, e.g., a level in anunaffected subject or a subject who is not at risk of developing PDAC,and/or a disease reference that represents a level of SIRT6 and Lin28bassociated with (aggressive) PDAC.

The predetermined level can be a single cut-off (threshold) value, suchas a median or mean, or a level that defines the boundaries of an upperor lower quartile, tertile, or other segment of a clinical trialpopulation that is determined to be statistically different from theother segments. It can be a range of cut-off (or threshold) values, suchas a confidence interval. It can be established based upon comparativegroups, such as where association with risk of developing PDAC orpresence of PDAC in one defined group is a fold higher, or lower, (e.g.,approximately 2-fold, 4-fold, 8-fold, 16-fold or more) than the risk orpresence of PDAC in another defined group. It can be a range, forexample, where a population of subjects (e.g., control subjects) isdivided equally (or unequally) into groups, such as a low-risk group, amedium-risk group and a high-risk group, or into quartiles, the lowestquartile being subjects with the lowest risk and the highest quartilebeing subjects with the highest risk, or into n-quantiles (i.e., nregularly spaced intervals) the lowest of the n-quantiles being subjectswith the lowest risk and the highest of the n-quantiles being subjectswith the highest risk.

In some embodiments, the predetermined level is a level or occurrence inthe same subject, e.g., at a different time point, e.g., an earlier timepoint.

Subjects associated with predetermined values are typically referred toas reference subjects. For example, in some embodiments, a controlreference subject does not have (aggressive) PDAC. In some cases it maybe desirable that the control subject has (aggressive) PDAC. A diseasereference subject is one who has or has an increased risk of developing(aggressive) PDAC. An increased risk is defined as a risk above the riskof subjects in the general population.

Thus, in some cases, the level of Lin28b in a subject being greater thanor equal to a reference level of Lin28b is indicative of PDAC oraggressive PDAC. In other cases, the level of Lin28b in a subject beingless than or equal to the reference level of Lin28b is indicative of theabsence of disease or normal risk of PDAC. In some embodiments, theamount by which the level in the subject is the greater than thereference level is sufficient to distinguish a subject from a controlsubject, and optionally is a statistically significantly greater thanthe level in a control subject. In cases where the level of Lin28b in asubject being equal to the reference level of Lin28b, the “being equal”refers to being approximately equal (e.g., not statistically different).

In some cases, the level of SIRT6 in a subject being less than or equalto a reference level of SIRT6 is indicative of PDAC or aggressive PDAC.In other cases, the level of SIRT6 in a subject being greater than orequal to the reference level of SIRT6 is indicative of the absence ofdisease or normal risk of PDAC. In some embodiments, the amount by whichthe level in the subject is the greater than the reference level issufficient to distinguish a subject from a control subject, andoptionally is a statistically significantly greater than the level in acontrol subject. In cases where the level of SIRT6 in a subject beingequal to the reference level of Lin28b, the “being equal” refers tobeing approximately equal (e.g., not statistically different).

The predetermined value can depend upon the particular population ofsubjects (e.g., human subjects) selected. For example, an apparentlyhealthy population will have a different ‘normal’ range of levels ofLin28b than will a population of subjects which have, or are likely tohave, PDAC. Accordingly, the predetermined values selected may take intoaccount the category (e.g., sex, age, health, risk, presence of otherdiseases) in which a subject (e.g., human subject) falls. Appropriateranges and categories can be selected with no more than routineexperimentation by those of ordinary skill in the art.

In characterizing likelihood, or risk, numerous predetermined values canbe established.

The methods described herein are useful for diagnosing and/or treatingPDAC, e.g., aggressive PDAC. In some embodiments, once it has beendetermined that a person has PDAC, or has an increased risk ofdeveloping PDAC, then a treatment, e.g., an inhibitory nucleic acid asdescribed herein, can optionally be administered.

Subjects to be Treated

In one aspect of the methods described herein, a subject is selected onthe basis that they have, or are at risk of developing, (aggressive)PDAC, e.g., a subject with a level of SIRT6 expression below a referencelevel and/or a level of Lin28b expression above a reference level. PDACdevelops from cells lining the ducts that carry the digestive juicesinto the main pancreatic duct and then on into the duodenum. They cangrow anywhere in the pancreas, although most often they are found in thehead of the pancreas. There are several very rare variants of PDAC,including adenosquamous carcinoma and colloid carcinoma.

A subject that has, or is at risk of developing, (aggressive) PDAC isone having one or more symptoms of the condition. Symptoms of(aggressive) PDAC are known to those of skill in the art and include,without limitation, abdominal pain, jaundice, weight loss, bowelproblems, e.g., steatorrhea and diarrhea, nausea, vomiting, indigestion,heartburn, fever, shivering, diabetes, back pain, extremetiredness/fatigue, feeling unusually full after food, venousthromboembolism, and unexplained acute pancreatitis. There is evidencethat age, smoking, being overweight, a family history of pancreaticcancer, pancreatitis, diabetes, alcohol, red and processed meat, historyof cancer, blood group A, hepatitis, stomach or gall bladder surgery,and Helicobacter pylori infection may increase risk of pancreaticcancer.

The methods are effective for a variety of subjects including mammals,e.g., humans and other animals, such as laboratory animals, e.g., mice,rats, rabbits, or monkeys, or domesticated and farm animals, e.g., cats,dogs, goats, sheep, pigs, cows, or horses.

Methods of Modulating Gene Expression

The methods described herein can be used for modulating expression ofoncogenes and tumor suppressors in cells, e.g., PDAC cells. For example,to decrease expression of LIN28B in a cell, the methods includeintroducing into the cell an inhibitory nucleic acid or small moleculethat specifically binds, or is complementary, to LIN28B mRNA.

In preferred embodiments, the inhibitory nucleic acid binds to a regionwithin or near (e.g., within 100, 200, 300, 400, 500, 600, 700, 1K, 2K,or 5K bases of) LIN28B. A nucleic acid that binds “specifically” bindsprimarily to the target LIN28B RNA to inhibit Lin28b but not of othernon-target RNAs. The specificity of the nucleic acid interaction thusrefers to its function (e.g., inhibiting Lin28b gene expression) ratherthan its hybridization capacity. Inhibitory nucleic acids may exhibitnonspecific binding to other sites in the genome or other RNAs, withoutinterfering with binding of other regulatory proteins and withoutcausing degradation of the non-specifically-bound RNA. Thus, thisnonspecific binding does not significantly affect function of othernon-target RNAs and results in no significant adverse effects.

These methods can be used to treat (aggressive) PDAC in a subject byadministering to the subject a composition (e.g., as described herein)comprising an LIN28B-inhibitory nucleic acid.

As used herein, treating includes “prophylactic treatment,” which meansreducing the incidence of or preventing (or reducing risk of) a sign orsymptom of PDAC in a patient at risk for the disease, and “therapeutictreatment,” which means reducing signs or symptoms of PDAC, reducingprogression of PDAC, reducing severity of PDAC, in a patient diagnosedwith PDAC, e.g., inhibiting tumor cell proliferation, increasing tumorcell death or killing, inhibiting rate of tumor cell growth ormetastasis, reducing size of tumors, reducing number of tumors, reducingnumber of metastases, increasing 1-year or 5-year survival rate.

As used herein, the terms “cancer”, “hyperproliferative”, and“neoplastic” refer to cells having the capacity for autonomous growth,i.e., an abnormal state or condition characterized by rapidlyproliferating cell growth. Hyperproliferative and neoplastic diseasestates may be categorized as pathologic, i.e., characterizing orconstituting a disease state, or may be categorized as non-pathologic,i.e., a deviation from normal but not associated with a disease state.The term is meant to include all types of cancerous growths or oncogenicprocesses, metastatic tissues or malignantly transformed cells, tissues,or organs, irrespective of histopathologic type or stage ofinvasiveness. “Pathologic hyperproliferative” cells occur in diseasestates characterized by malignant tumor growth. Examples ofnon-pathologic hyperproliferative cells include proliferation of cellsassociated with wound repair.

The term “carcinoma” is art recognized and refers to malignancies ofepithelial or endocrine tissues including those forming from tissue ofthe pancreas. The term also includes carcinosarcomas, e.g., whichinclude malignant tumors composed of carcinomatous and sarcomatoustissues. An “adenocarcinoma” refers to a carcinoma derived fromglandular tissue or in which the tumor cells form recognizable glandularstructures.

In some embodiments, the methods described herein include administeringa composition, e.g., a sterile composition, comprising an inhibitorynucleic acid that is complementary to LIN28B. Inhibitory nucleic acidsfor use in practicing the methods described herein can be an antisenseor small interfering RNA, including but not limited to a shRNA or siRNA.In some embodiments, the inhibitory nucleic acid is a modified nucleicacid polymer (e.g., a locked nucleic acid (LNA) molecule). Inhibitorynucleic acids have been employed as therapeutic moieties in thetreatment of disease states in animals, including humans. Inhibitorynucleic acids can be useful therapeutic modalities that can beconfigured to be useful in treatment regimens for the treatment ofcells, tissues, and animals, especially humans.

For therapeutics, an animal, preferably a human, suspected of having(aggressive) PDAC is treated by administering an inhibitory nucleic acidin accordance with this invention. For example, in one non-limitingembodiment, the methods comprise a step of administering to the animalin need of treatment, a therapeutically effective amount of aninhibitory nucleic acid as described herein.

Inhibitory Nucleic Acids

Inhibitory nucleic acids useful in the present methods and compositionsinclude antisense oligonucleotides, ribozymes, external guide sequence(EGS) oligonucleotides, siRNA compounds, single- or double-stranded RNAinterference (RNAi) compounds such as siRNA compounds, moleculescomprising modified bases, locked nucleic acid molecules (LNAmolecules), antagomirs, peptide nucleic acid molecules (PNA molecules),and other oligomeric compounds or oligonucleotide mimetics whichhybridize to at least a portion of the target nucleic acid and modulateits function. In some embodiments, the inhibitory nucleic acids includeantisense RNA, antisense DNA, chimeric antisense oligonucleotides,antisense oligonucleotides comprising modified linkages, interferenceRNA (RNAi), short interfering RNA (siRNA); a micro, interfering RNA(miRNA); a small, temporal RNA (stRNA); or a short, hairpin RNA (shRNA);small RNA-induced gene activation (RNAa); small activating RNAs(saRNAs), or combinations thereof. See, e.g., WO 2010040112.

In the present methods, the inhibitory nucleic acids are preferablydesigned to target LIN28B. These “inhibitory” nucleic acids are believedto work by inhibiting expression of LIN28B.

In some embodiments, the inhibitory nucleic acids are 10 to 50, 13 to50, or 13 to 30 nucleotides in length. One having ordinary skill in theart will appreciate that this embodies oligonucleotides having antisense(complementary) portions of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length,or any range therewithin. It is understood that non-complementary basesmay be included in such inhibitory nucleic acids; for example, aninhibitory nucleic acid 30 nucleotides in length may have a portion of15 bases that is complementary to the targeted RNA. In some embodiments,the oligonucleotides are 15 nucleotides in length. In some embodiments,the antisense or oligonucleotide compounds of the invention are 12 or 13to 30 nucleotides in length. One having ordinary skill in the art willappreciate that this embodies inhibitory nucleic acids having antisense(complementary) portions of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length, or any rangetherewithin.

Preferably, the inhibitory nucleic acid comprises one or moremodifications comprising: a modified sugar moiety, and/or a modifiedinternucleoside linkage, and/or a modified nucleotide and/orcombinations thereof. It is not necessary for all positions in a givenoligonucleotide to be uniformly modified, and in fact more than one ofthe modifications described herein may be incorporated in a singleoligonucleotide or even at within a single nucleoside within anoligonucleotide.

In some embodiments, the inhibitory nucleic acids are chimericoligonucleotides that contain two or more chemically distinct regions,each made up of at least one nucleotide. These oligonucleotidestypically contain at least one region of modified nucleotides thatconfers one or more beneficial properties (such as, for example,increased nuclease resistance, increased uptake into cells, increasedbinding affinity for the target) and a region that is a substrate forenzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Chimericinhibitory nucleic acids of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides, and/or oligonucleotide mimetics as described above.Such compounds have also been referred to in the art as hybrids orgapmers. Representative United States patents that teach the preparationof such hybrid structures comprise, but are not limited to, U.S. Pat.Nos. 5,013,830; 5,149,797; 5, 220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,each of which is herein incorporated by reference.

In some embodiments, the inhibitory nucleic acid comprises at least onenucleotide modified at the 2′ position of the sugar, most preferably a2′-O-alkyl, 2′-O-alkyl-O-alkyl or 2′-fluoro-modified nucleotide. Inother preferred embodiments, RNA modifications include 2′-fluoro,2′-amino and 2′ O-methyl modifications on the ribose of pyrimidines,abasic residues or an inverted base at the 3′ end of the RNA. Suchmodifications are routinely incorporated into oligonucleotides and theseoligonucleotides have been shown to have a higher Tm (i.e., highertarget binding affinity) than; 2′-deoxyoligonucleotides against a giventarget.

A number of nucleotide and nucleoside modifications have been shown tomake the oligonucleotide into which they are incorporated more resistantto nuclease digestion than the native oligodeoxynucleotide; thesemodified oligos survive intact for a longer time than unmodifiedoligonucleotides. Specific examples of modified oligonucleotides includethose comprising modified backbones, for example, phosphorothioates,phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkylintersugar linkages or short chain heteroatomic or heterocyclicintersugar linkages. Most preferred are oligonucleotides withphosphorothioate backbones and those with heteroatom backbones,particularly CH₂—NH—O—CH₂, CH,˜N(CH₃)˜O˜CH₂ (known as amethylene(methylimino) or MMI backbone, CH₂—O—N (CH₃)—CH₂, CH₂—N (CH₃)—N(CH₃)—CH₂ and O—N (CH₃)—CH₂—CH₂ backbones, wherein the nativephosphodiester backbone is represented as O—P—O—CH,); amide backbones(see De Mesmaeker et al. Ace. Chem. Res. 1995, 28:366-374); morpholinobackbone structures (see Summerton and Weller, U.S. Pat. No. 5,034,506);peptide nucleic acid (PNA) backbone (wherein the phosphodiester backboneof the oligonucleotide is replaced with a polyamide backbone, thenucleotides being bound directly or indirectly to the aza nitrogen atomsof the polyamide backbone, see Nielsen et al., Science 1991, 254, 1497).Phosphorus-containing linkages include, but are not limited to,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates comprising 3′alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates comprising 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′; see U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5, 177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455, 233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563, 253; 5,571,799;5,587,361; and 5,625,050.

Morpholino-based oligomeric compounds are described in Dwaine A. Braaschand David R. Corey, Biochemistry, 2002, 41(14), 4503-4510); Genesis,volume 30, issue 3, 2001; Heasman, J., Dev. Biol., 2002, 243, 209-214;Nasevicius et al., Nat. Genet., 2000, 26, 216-220; Lacerra et al., Proc.Natl. Acad. Sci., 2000, 97, 9591-9596; and U.S. Pat. No. 5,034,506. Insome embodiments, the morpholino-based oligomeric compound is aphosphorodiamidate morpholino oligomer (PMO) (e.g., as described inIverson, Curr. Opin. Mol. Ther., 3:235-238, 2001; and Wang et al., J.Gene Med., 12:354-364, 2010; the disclosures of which are incorporatedherein by reference in their entireties.

Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wanget al., J. Am. Chem. Soc., 2000, 122, 8595-8602.

Modified oligonucleotide backbones that do not include a phosphorus atomtherein have backbones that are formed by short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatom and alkyl orcycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These comprisethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S, and CH2 component parts; see U.S. Pat. Nos. 5,034,506; 5,166,315;5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564;5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307;5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and5,677,439, each of which is herein incorporated by reference.

Modified oligonucleotides are also known that include oligonucleotidesthat are based on or constructed from arabinonucleotide or modifiedarabinonucleotide residues. Arabinonucleosides are stereoisomers ofribonucleosides, differing only in the configuration at the 2′-positionof the sugar ring. In some embodiments, a 2′-arabino modification is2′-F arabino. In some embodiments, the modified oligonucleotide is2′-fluoro-D-arabinonucleic acid (FANA) (as described in, for example,Lon et al., Biochem., 41:3457-3467, 2002 and Min et al., Bioorg. Med.Chem. Lett., 12:2651-2654, 2002; the disclosures of which areincorporated herein by reference in their entireties). Similarmodifications can also be made at other positions on the sugar,particularly the 3′ position of the sugar on a 3′ terminal nucleoside orin 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminalnucleotide.

PCT Publication No. WO 99/67378 discloses arabinonucleic acids (ANA)oligomers and their analogues for improved sequence specific inhibitionof gene expression via association to complementary messenger RNA.

Other preferred modifications include ethylene-bridged nucleic acids(ENAs) (e.g., International Patent Publication No. WO 2005/042777,Morita et al., Nucleic Acid Res., Suppl 1:241-242, 2001; Surono et al.,Hum. Gene Ther., 15:749-757, 2004; Koizumi, Curr. Opin. Mol. Ther.,8:144-149, 2006 and Horie et al., Nucleic Acids Symp. Ser (Oxf),49:171-172, 2005; the disclosures of which are incorporated herein byreference in their entireties). Preferred ENAs include, but are notlimited to, 2′-O,4′-C-ethylene-bridged nucleic acids.

Examples of LNAs are described in WO 2008/043753 and include compoundsof the following formula.

where X and Y are independently selected among the groups —O—,

—S—, —N(H)—, N(R)—, —CH2— or —CH— (if part of a double bond),

—CH₂—O—, —CH₂—S—, —CH₂—N(H)—, —CH₂—N(R)—, —CH₂—CH₂— or —CH₂—CH— (if partof a double bond),

—CH═CH—, where R is selected from hydrogen and C₁₋₄-alkyl; Z and Z* areindependently selected among an internucleoside linkage, a terminalgroup or a protecting group; B constitutes a natural or non-naturalnucleotide base moiety; and the asymmetric groups may be found in eitherorientation.

Preferably, the LNA used in the oligomer of the invention comprises atleast one LNA unit according any of the formulas

wherein Y is —O—, —S—, —NH—, or N(R^(H)); Z and Z* are independentlyselected among an internucleoside linkage, a terminal group or aprotecting group; B constitutes a natural or non-natural nucleotide basemoiety, and RH is selected from hydrogen and C₁₋₄-alkyl.

Preferably, the LNA used in the oligomeric compound, such as anantisense oligonucleotide, of the invention comprises at least onenucleotide comprises a LNA unit according any of the formulas shown in“Scheme 2” of PCT/DK2006/000512.

Preferably, the LNA used in the oligomer of the invention comprisesinternucleoside linkages selected from —O—P(O)₂—O—, —O—P(O,S)—O—,—O—P(S)₂—O—, —S—P(O)₂—O—, —S—P(O,S)—O—, —S-P(S)₂—O—, —O—(O)₂—S—,—O—P(O,S)—S—, —S—P(O)₂—S—, —O—PO(R^(H))—O—, O—PO(OCH₃)—O—,—O—PO(NR^(H))—O—, —O—PO(OCH₂CH₂S—R)—O—, —O—PO(BH₃)—O—,—O—PO(NHR^(H))—O—, —O—P(O)₂—NR^(H)—, —NR^(H)—P(O)₂—O—, —NR^(H)—CO—O—,where R^(H) is selected from hydrogen and C₁₋₄-alkyl.

Specifically, preferred LNA units are shown in Scheme 1:

The term “thio-LNA” comprises a locked nucleotide in which at least oneof X or Y in the general formula above is selected from S or —CH2-S—.Thio-LNA can be in both beta-D and alpha-L-configuration.

The term “amino-LNA” comprises a locked nucleotide in which at least oneof X or Y in the general formula above is selected from —N(H)—, N(R)—,CH₂—N(H)—, and —CH₂—N(R)— where R is selected from hydrogen andC₁₋₄-alkyl. Amino-LNA can be in both beta-D and alpha-L-configuration.

The term “oxy-LNA” comprises a locked nucleotide in which at least oneof X or Y in the general formula above represents —O— or —CH₂—O—.Oxy-LNA can be in both beta-D and alpha-L-configuration.

The term “ena-LNA” comprises a locked nucleotide in which Y in thegeneral formula above is —CH₂—O— (where the oxygen atom of —CH₂—O— isattached to the 2′-position relative to the base B). LNAs are describedin additional detail below.

One or more substituted sugar moieties can also be included, e.g., oneof the following at the 2′ position: OH, SH, SCH₃, F, OCN, OCH₃, OCH₃O(CH₂)n CH₃, O(CH₂)n NH₂ or O(CH₂)n CH₃ where n is from 1 to about 10;Ci to C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl oraralkyl; Cl; Br; CN; CF3 ; OCF3; O—, S—, or N-alkyl; O—, S—, orN-alkenyl; SOCH3; SO2 CH3; ONO2; NO2; N3; NH2; heterocycloalkyl;heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl;an RNA cleaving group; a reporter group; an intercalator; a group forimproving the pharmacokinetic properties of an oligonucleotide; or agroup for improving the pharmacodynamic properties of an oligonucleotideand other substituents having similar properties. A preferredmodification includes 2′-methoxyethoxy [2′-0-CH₂CH₂OCH₃, also known as2′-O-(2-methoxyethyl)] (Martin et al, Hely. Chim. Acta, 1995, 78, 486).Other preferred modifications include 2′-methoxy (2′-0-CH₃), 2′-propoxy(2′-OCH₂ CH₂CH₃) and 2′-fluoro (2′-F). Similar modifications may also bemade at other positions on the oligonucleotide, particularly the 3′position of the sugar on the 3′ terminal nucleotide and the 5′ positionof 5′ terminal nucleotide. Oligonucleotides may also have sugar mimeticssuch as cyclobutyls in place of the pentofuranosyl group.

Inhibitory nucleic acids can also include, additionally oralternatively, nucleobase (often referred to in the art simply as“base”) modifications or substitutions. As used herein, “unmodified” or“natural” nucleobases include adenine (A), guanine (G), thymine (T),cytosine (C) and uracil (U). Modified nucleobases include nucleobasesfound only infrequently or transiently in natural nucleic acids, e.g.,hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly5-methylcytosine (also referred to as 5-methyl-2′ deoxycytosine andoften referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC),glycosyl HMC and gentobiosyl HMC, isocytosine, pseudoisocytosine, aswell as synthetic nucleobases, e.g., 2-aminoadenine,2-(methylamino)adenine, 2-(imidazolylalkyl)adenine,2-(aminoalklyamino)adenine or other heterosubstituted alkyladenines,2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil,5-propynyluracil, 8-azaguanine, 7-deazaguanine, N6(6-aminohexyl)adenine, 6-aminopurine, 2-aminopurine,2-chloro-6-aminopurine and 2,6-diaminopurine or other diaminopurines.See, e.g., Kornberg, “DNA Replication,” W. H. Freeman & Co., SanFrancisco, 1980, pp75-77; and Gebeyehu, G., et al. Nucl. Acids Res.,15:4513 (1987)). A “universal” base known in the art, e.g., inosine, canalso be included. 5-Me-C substitutions have been shown to increasenucleic acid duplex stability by 0.6-1.2<0>C. (Sanghvi, in Crooke, andLebleu, eds., Antisense Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are presently preferred basesubstitutions.

It is not necessary for all positions in a given oligonucleotide to beuniformly modified, and in fact more than one of the modificationsdescribed herein may be incorporated in a single oligonucleotide or evenat within a single nucleoside within an oligonucleotide.

In some embodiments, both a sugar and an internucleoside linkage, i.e.,the backbone, of the nucleotide units are replaced with novel groups.The base units are maintained for hybridization with an appropriatenucleic acid target compound. One such oligomeric compound, anoligonucleotide mimetic that has been shown to have excellenthybridization properties, is referred to as a peptide nucleic acid(PNA). In PNA compounds, the sugar-backbone of an oligonucleotide isreplaced with an amide containing backbone, for example, anaminoethylglycine backbone. The nucleobases are retained and are bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone. Representative United States patents that teach thepreparation of PNA compounds include, but are not limited to, U.S. Pat.Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

Inhibitory nucleic acids can also include one or more nucleobase (oftenreferred to in the art simply as “base”) modifications or substitutions.As used herein, “unmodified” or “natural” nucleobases comprise thepurine bases adenine (A) and guanine (G), and the pyrimidine basesthymine (T), cytosine (C) and uracil (U). Modified nucleobases compriseother synthetic and natural nucleobases such as 5-methylcytosine(5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine,2-aminoadenine, 6-methyl and other alkyl derivatives of adenine andguanine, 2-propyl and other alkyl derivatives of adenine and guanine,2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil andcytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil (pseudo-uracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylquanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

Further, nucleobases comprise those disclosed in U.S. Pat. No.3,687,808, those disclosed in “The Concise Encyclopedia of PolymerScience And Engineering”, pages 858-859, Kroschwitz, ed. John Wiley &Sons, 1990;, those disclosed by Englisch et al., Angewandle Chemie,International Edition, 1991, 30, page 613, and those disclosed bySanghvi, Chapter 15, Antisense Research and Applications,” pages289-302, Crooke, and Lebleu, eds., CRC Press, 1993. Certain of thesenucleobases are particularly useful for increasing the binding affinityof the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, comprising 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2<0>C (Sanghvi, etal., eds, “Antisense Research and Applications,” CRC Press, Boca Raton,1993, pp. 276-278) and are presently preferred base substitutions, evenmore particularly when combined with 2′-O-methoxyethyl sugarmodifications. Modified nucleobases are described in U.S. Pat. Nos.3,687,808, as well as 4,845,205; 5,130,302; 5,134,066; 5,175,273;5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177;5,525,711; 5,552,540; 5,587,469; 5,596,091; 5,614,617; 5,750,692; and5,681,941, each of which is herein incorporated by reference.

In some embodiments, the inhibitory nucleic acids are chemically linkedto one or more moieties or conjugates that enhance the activity,cellular distribution, or cellular uptake of the oligonucleotide. Forexample, one or more inhibitory nucleic acids, of the same or differenttypes, can be conjugated to each other; or inhibitory nucleic acids canbe conjugated to targeting moieties with enhanced specificity for a celltype or tissue type. Such moieties include, but are not limited to,lipid moieties such as a cholesterol moiety (Letsinger et al., Proc.Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan etal., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g.,hexyl-S-tritylthiol (Manoharan et al, Ann. N. Y. Acad. Sci., 1992, 660,306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770),a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20,533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues(Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al.,Biochimie, 1993, 75, 49-54), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Mancharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-toxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996,277, 923-937). See also U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105;5,525,465; 5,541,313; 5,545,730; 5,552, 538; 5,578,717, 5,580,731;5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025;4,762, 779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582;4,958,013; 5,082, 830; 5,112,963; 5,214,136; 5,082,830; 5,112,963;5,214,136; 5, 245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250;5,292,873; 5,317,098; 5,371,241, 5,391, 723; 5,416,203, 5,451,463;5,510,475; 5,512,667; 5,514,785; 5, 565,552; 5,567,810; 5,574,142;5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599, 928 and5,688,941, each of which is herein incorporated by reference.

These moieties or conjugates can include conjugate groups covalentlybound to functional groups such as primary or secondary hydroxyl groups.Conjugate groups of the invention include intercalators, reportermolecules, polyamines, polyamides, polyethylene glycols, polyethers,groups that enhance the pharmacodynamic properties of oligomers, andgroups that enhance the pharmacokinetic properties of oligomers. Typicalconjugate groups include cholesterols, lipids, phospholipids, biotin,phenazine, folate, phenanthridine, anthraquinone, acridine,fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance thepharmacodynamic properties, in the context of this invention, includegroups that improve uptake, enhance resistance to degradation, and/orstrengthen sequence-specific hybridization with the target nucleic acid.Groups that enhance the pharmacokinetic properties, in the context ofthis invention, include groups that improve uptake, distribution,metabolism or excretion of the compounds of the present invention.Representative conjugate groups are disclosed in International PatentApplication No. PCT/US92/09196, filed Oct. 23, 1992, and U.S. Pat. No.6,287,860, which are incorporated herein by reference. Conjugatemoieties include, but are not limited to, lipid moieties such as acholesterol moiety, cholic acid, a thioether, e.g., hexyl-5-tritylthiol,a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecylresidues, a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, apolyamine or a polyethylene glycol chain, or adamantane acetic acid, apalmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety. See, e.g., U.S. Pat. Nos. 4,828,979; 4,948,882;5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045;5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044;4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263;4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136;5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506;5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723;5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552;5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696;5,599,923; 5,599,928; and 5,688,941.

The inhibitory nucleic acids useful in the present methods aresufficiently complementary to the target RNA, e.g., hybridizesufficiently well and with sufficient biological functional specificity,to give the desired effect. “Complementary” refers to the capacity forpairing, through base stacking and specific hydrogen bonding, betweentwo sequences comprising naturally or non-naturally occurring (e.g.,modified as described above) bases (nucleosides) or analogs thereof. Forexample, if a base at one position of an inhibitory nucleic acid iscapable of hydrogen bonding with a base at the corresponding position ofan RNA, then the bases are considered to be complementary to each otherat that position. 100% complementarity is not required. As noted above,inhibitory nucleic acids can comprise universal bases, or inert abasicspacers that provide no positive or negative contribution to hydrogenbonding. Base pairings may include both canonical Watson-Crick basepairing and non-Watson-Crick base pairing (e.g., Wobble base pairing andHoogsteen base pairing). It is understood that for complementary basepairings, adenosine-type bases (A) are complementary to thymidine-typebases (T) or uracil-type bases (U), that cytosine-type bases (C) arecomplementary to guanosine-type bases (G), and that universal bases suchas such as 3-nitropyrrole or 5-nitroindole can hybridize to and areconsidered complementary to any A, C, U, or T. Nichols et al., Nature,1994; 369:492-493 and Loakes et al., Nucleic Acids Res., 1994;22:4039-4043. Inosine (I) has also been considered in the art to be auniversal base and is considered complementary to any A, C, U, or T. SeeWatkins and SantaLucia, Nucl. Acids Research, 2005; 33 (19): 6258-6267.

In some embodiments, the location on a target RNA to which an inhibitorynucleic acids hybridizes is defined as a region to which a proteinbinding partner binds. Routine methods can be used to design aninhibitory nucleic acid that binds to this sequence with sufficientspecificity. In some embodiments, the methods include usingbioinformatics methods known in the art to identify regions of secondarystructure, e.g., one, two, or more stem-loop structures, or pseudoknots,and selecting those regions to target with an inhibitory nucleic acid.For example, methods of designing oligonucleotides similar to theinhibitory nucleic acids described herein, and various options formodified chemistries or formats, are exemplified in Lennox and Behlke,Gene Therapy (2011) 18:1111-1120, which is incorporated herein byreference in its entirety, with the understanding that the presentdisclosure does not target miRNA ‘seed regions’.

While the specific sequences of certain exemplary target segments areset forth herein, one of skill in the art will recognize that theseserve to illustrate and describe particular embodiments within the scopeof the present invention. Additional target segments are readilyidentifiable by one having ordinary skill in the art in view of thisdisclosure. Target segments 5-500 nucleotides in length comprising astretch of at least five (5) consecutive nucleotides within the proteinbinding region, or immediately adjacent thereto, are considered to besuitable for targeting as well. Target segments can include sequencesthat comprise at least the 5 consecutive nucleotides from the5′-terminus of one of the protein binding regions (the remainingnucleotides being a consecutive stretch of the same RNA beginningimmediately upstream of the 5′-terminus of the binding segment andcontinuing until the inhibitory nucleic acid contains about 5 to about100 nucleotides). Similarly preferred target segments are represented byRNA sequences that comprise at least the 5 consecutive nucleotides fromthe 3′-terminus of one of the illustrative preferred target segments(the remaining nucleotides being a consecutive stretch of the same RNAbeginning immediately downstream of the 3′-terminus of the targetsegment and continuing until the inhibitory nucleic acid contains about5 to about 100 nucleotides). One having skill in the art armed with thesequences provided herein will be able, without undue experimentation,to identify further preferred protein binding regions to target withcomplementary inhibitory nucleic acids.

In the context of the present disclosure, hybridization means basestacking and hydrogen bonding, which may be Watson-Crick, Hoogsteen orreversed Hoogsteen hydrogen bonding, between complementary nucleoside ornucleotide bases. For example, adenine and thymine are complementarynucleobases which pair through the formation of hydrogen bonds.Complementary, as the term is used in the art, refers to the capacityfor precise pairing between two nucleotides. For example, if anucleotide at a certain position of an oligonucleotide is capable ofhydrogen bonding with a nucleotide at the same position of a RNAmolecule, then the inhibitory nucleic acid and the RNA are considered tobe complementary to each other at that position. The inhibitory nucleicacids and the RNA are complementary to each other when a sufficientnumber of corresponding positions in each molecule are occupied bynucleotides that can hydrogen bond with each other through their bases.Thus, “specifically hybridizable” and “complementary” are terms whichare used to indicate a sufficient degree of complementarity or precisepairing such that stable and specific binding occurs between theinhibitory nucleic acid and the RNA target. For example, if a base atone position of an inhibitory nucleic acid is capable of hydrogenbonding with a base at the corresponding position of a RNA, then thebases are considered to be complementary to each other at that position.100% complementarity is not required.

It is understood in the art that a complementary nucleic acid sequenceneed not be 100% complementary to that of its target nucleic acid to bespecifically hybridizable. A complementary nucleic acid sequence forpurposes of the present methods is specifically hybridizable whenbinding of the sequence to the target RNA molecule interferes with thenormal function of the target RNA to cause a loss of activity (e.g.,inhibiting LIN28B) and there is a sufficient degree of complementarityto avoid non-specific binding of the sequence to non-target RNAsequences under conditions in which avoidance of the non-specificbinding is desired, e.g., under physiological conditions in the case ofin vivo assays or therapeutic treatment, and in the case of in vitroassays, under conditions in which the assays are performed undersuitable conditions of stringency. For example, stringent saltconcentration will ordinarily be less than about 750 mM NaCl and 75 mMtrisodium citrate, preferably less than about 500 mM NaCl and 50 mMtrisodium citrate, and more preferably less than about 250 mM NaCl and25 mM trisodium citrate. Low stringency hybridization can be obtained inthe absence of organic solvent, e.g., formamide, while high stringencyhybridization can be obtained in the presence of at least about 35%formamide, and more preferably at least about 50% formamide. Stringenttemperature conditions will ordinarily include temperatures of at leastabout 30° C., more preferably of at least about 37° C., and mostpreferably of at least about 42° C. Varying additional parameters, suchas hybridization time, the concentration of detergent, e.g., sodiumdodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA,are well known to those skilled in the art. Various levels of stringencyare accomplished by combining these various conditions as needed. In apreferred embodiment, hybridization will occur at 30° C. in 750 mM NaCl,75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment,hybridization will occur at 37° C. in 500 mM NaCl, 50 mM trisodiumcitrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA(ssDNA). In a most preferred embodiment, hybridization will occur at 42°C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and200 μg/ml ssDNA. Useful variations on these conditions will be readilyapparent to those skilled in the art.

For most applications, washing steps that follow hybridization will alsovary in stringency. Wash stringency conditions can be defined by saltconcentration and by temperature. As above, wash stringency can beincreased by decreasing salt concentration or by increasing temperature.For example, stringent salt concentration for the wash steps willpreferably be less than about 30 mM NaCl and 3 mM trisodium citrate, andmost preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.Stringent temperature conditions for the wash steps will ordinarilyinclude a temperature of at least about 25° C., more preferably of atleast about 42° C., and even more preferably of at least about 68° C. Ina preferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, washsteps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and0.1% SDS. In a more preferred embodiment, wash steps will occur at 68°C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additionalvariations on these conditions will be readily apparent to those skilledin the art. Hybridization techniques are well known to those skilled inthe art and are described, for example, in Benton and Davis (Science196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology,Wiley Interscience, New York, 2001); Berger and Kimmel (Guide toMolecular Cloning Techniques, 1987, Academic Press, New York); andSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, New York.

In general, the inhibitory nucleic acids useful in the methods describedherein have at least 80% sequence complementarity to a target regionwithin the target nucleic acid, e.g., 90%, 95%, or 100% sequencecomplementarity to the target region within an RNA. For example, anantisense compound in which 18 of 20 nucleobases of the antisenseoligonucleotide are complementary, and would therefore specificallyhybridize, to a target region would represent 90 percentcomplementarity. Percent complementarity of an inhibitory nucleic acidwith a region of a target nucleic acid can be determined routinely usingbasic local alignment search tools (BLAST programs) (Altschul et al., J.Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7,649-656). Antisense and other compounds of the invention that hybridizeto an RNA are identified through routine experimentation. In general theinhibitory nucleic acids must retain specificity for their target, i.e.,either do not directly bind to, or do not directly significantly affectexpression levels of, transcripts other than the intended target.

Target-specific effects, with corresponding target-specific functionalbiological effects, are possible even when the inhibitory nucleic acidexhibits non-specific binding to a large number of non-target RNAs. Forexample, short 8 base long inhibitory nucleic acids that are fullycomplementary to a RNA may have multiple 100% matches to hundreds ofsequences in the genome, yet may produce target-specific effects, e.g.downregulation of a MIN28B. 8-base inhibitory nucleic acids have beenreported to prevent exon skipping with a high degree of specificity andreduced off-target effect. See Singh et al., RNA Biol., 2009; 6(3):341-350. 8-base inhibitory nucleic acids have been reported to interferewith miRNA activity without significant off-target effects. See Obad etal., Nature Genetics, 2011; 43: 371-378.

For further disclosure regarding inhibitory nucleic acids, please seeUS2010/0317718 (antisense oligos); US2010/0249052 (double-strandedribonucleic acid (dsRNA)); US2009/0181914 and US2010/0234451 (LNAmolecules); US2007/0191294 (siRNA analogues); US2008/0249039 (modifiedsiRNA); and WO2010/129746 and WO2010/040112 (inhibitory nucleic acids).

Antisense

In some embodiments, the inhibitory nucleic acids are antisenseoligonucleotides. Antisense oligonucleotides are typically designed toblock expression of a DNA or RNA target by binding to the target andhalting expression at the level of transcription, translation, orsplicing. Antisense oligonucleotides of the present invention arecomplementary nucleic acid sequences designed to hybridize understringent conditions to an RNA in vitro, and are expected to inhibit theactivity of Lin28b in vivo. Thus, oligonucleotides are chosen that aresufficiently complementary to the target, i.e., that hybridizesufficiently well and with sufficient biological functional specificity,to give the desired effect.

Modified Base, including Locked Nucleic Acids (LNAs)

In some embodiments, the inhibitory nucleic acids used in the methodsdescribed herein comprise one or more modified bonds or bases. Modifiedbases include phosphorothioate, methylphosphonate, peptide nucleicacids, or locked nucleic acids (LNAs). Preferably, the modifiednucleotides are part of locked nucleic acid molecules, including[alpha]-L-LNAs. LNAs include ribonucleic acid analogues wherein theribose ring is “locked” by a methylene bridge between the 2′-oxgygen andthe 4′-carbon—i.e., oligonucleotides containing at least one LNAmonomer, that is, one 2′-O,4′-C-methylene-β-D-ribofuranosyl nucleotide.LNA bases form standard Watson-Crick base pairs but the lockedconfiguration increases the rate and stability of the basepairingreaction (Jepsen et al., Oligonucleotides, 14, 130-146 (2004)). LNAsalso have increased affinity to base pair with RNA as compared to DNA.These properties render LNAs especially useful as probes forfluorescence in situ hybridization (FISH) and comparative genomichybridization, as knockdown tools for miRNAs, and as antisenseoligonucleotides to target mRNAs or other RNAs, e.g., RNAs as describedherein.

The modified base/LNA molecules can include molecules comprising 10-30,e.g., 12-24, e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, or 30 nucleotides in each strand, wherein one of thestrands is substantially identical, e.g., at least 80% (or more, e.g.,85%, 90%, 95%, or 100%) identical, e.g., having 3, 2, 1, or 0 mismatchednucleotide(s), to a target region in the RNA. The modified base/LNAmolecules can be chemically synthesized using methods known in the art.

The modified base/LNA molecules can be designed using any method knownin the art; a number of algorithms are known, and are commerciallyavailable (e.g., on the internet, for example at exiqon.com). See, e.g.,You et al., Nucl. Acids. Res. 34:e60 (2006); McTigue et al.,Biochemistry 43:5388-405 (2004); and Levin et al., Nucl. Acids. Res.34:e142 (2006). For example, “gene walk” methods, similar to those usedto design antisense oligos, can be used to optimize the inhibitoryactivity of a modified base/LNA molecule; for example, a series ofoligonucleotides of 10-30 nucleotides spanning the length of a targetRNA can be prepared, followed by testing for activity. Optionally, gaps,e.g., of 5-10 nucleotides or more, can be left between the LNAs toreduce the number of oligonucleotides synthesized and tested. GC contentis preferably between about 30-60%. General guidelines for designingmodified base/LNA molecules are known in the art; for example, LNAsequences will bind very tightly to other LNA sequences, so it ispreferable to avoid significant complementarity within an LNA molecule.Contiguous runs of three or more Gs or Cs, or more than four LNAresidues, should be avoided where possible (for example, it may not bepossible with very short (e.g., about 9-10 nt) oligonucleotides). Insome embodiments, the LNAs are xylo-LNAs.

For additional information regarding LNA molecules see U.S. Pat. Nos.6,268,490; 6,734,291; 6,770,748; 6,794,499; 7,034,133; 7,053,207;7,060,809; 7,084,125; and 7,572,582; and U.S. Pre-Grant Pub. Nos.20100267018; 20100261175; and 20100035968; Koshkin et al. Tetrahedron54,3607-3630 (1998); Obika et al. Tetrahedron Lett. 39,5401-5404 (1998);Jepsen et al., Oligonucleotides 14:130-146 (2004); Kauppinen et al.,Drug Disc. Today 2(3):287-290 (2005); and Ponting et al., Cell136(4):629-641 (2009), and references cited therein.

As demonstrated herein and previously (see, e.g., WO 2012/065143 and WO2012/087983, incorporated herein by reference), LNA molecules can beused as a valuable tool to manipulate and aid analysis of RNAs.Advantages offered by an LNA molecule-based system are the relativelylow costs, easy delivery, and rapid action. While other inhibitorynucleic acids may exhibit effects after longer periods of time, LNAmolecules exhibit effects that are more rapid, e.g., a comparativelyearly onset of activity, are fully reversible after a recovery periodfollowing the synthesis of new RNA, and occur without causingsubstantial or substantially complete RNA cleavage or degradation. Oneor more of these design properties may be desired properties of theinhibitory nucleic acids of the invention. Additionally, LNA moleculesmake possible the systematic targeting of domains within much longernuclear transcripts. Although a PNA-based system has been describedearlier, the effects on Xi were apparent only after 24 hours (Beletskiiet al., Proc Natl Acad Sci USA. 2001; 98:9215-9220). The LNA technologyenables high-throughput screens for functional analysis of non-codingRNAs and also provides a novel tool to manipulate chromatin states invivo for therapeutic applications.

In various related aspects, the methods described herein include usingLNA molecules to target RNAs for a number of uses, including as aresearch tool to probe the function of a specific RNA, e.g., in vitro orin vivo. The methods include selecting one or more desired RNAs,designing one or more LNA molecules that target the RNA, providing thedesigned LNA molecule, and administering the LNA molecule to a cell oranimal. The methods can optionally include selecting a region of the RNAand designing one or more LNA molecules that target that region of theRNA.

Aberrant imprinted gene expression is implicated in several diseasesincluding Long QT syndrome, Beckwith-Wiedemann, Prader-Willi, andAngelman syndromes, as well as behavioral disorders and carcinogenesis(see, e.g., Falls et al., Am. J. Pathol. 154:635-647 (1999); Lalande,Annu Rev Genet 30:173-195 (1996); Hall, Ann Rev Med. 48:35-44 (1997)).LNA molecules can be created to treat such imprinted diseases. As oneexample, the long QT Syndrome can be caused by a K⁻ gatedcalcium-channel encoded by Kcnql. This gene is regulated by itsantisense counterpart, the long noncoding RNA, Kcnqlotl (Pandey et al.,Mol Cell. 2008 Oct. 24; 32(2):232-46). Disease arises when Kcnqlotl isaberrantly expressed. LNA molecules can be created to downregulateKcnqlotl, thereby restoring expression of Kcnql. As another example, LNAmolecules could inhibit RNA cofactors for polycomb complex chromatinmodifiers to reverse the imprinted defect.

From a commercial and clinical perspective, the timepoints between about1 to 24 hours potentially define a window for epigenetic reprogramming.The advantage of the LNA system is that it works quickly, with a definedhalf-life, and is therefore reversible upon degradation of LNAs, at thesame time that it provides a discrete timeframe during which epigeneticmanipulations can be made. By targeting nuclear long RNAs, LNA moleculesor similar polymers, e.g., xylo-LNAs, might be utilized to manipulatethe chromatin state of cells in culture or in vivo, by transientlyeliminating the regulatory RNA and associated proteins long enough toalter the underlying locus for therapeutic purposes. In particular, LNAmolecules or similar polymers that specifically bind to, or arecomplementary to, LIN28B can inhibit LIN28B expression, in agene-specific fashion.

Interfering RNA, including siRNA/shRNA

In some embodiments, the inhibitory nucleic acid sequence that iscomplementary to an RNA can be an interfering RNA, including but notlimited to a small interfering RNA (“siRNA”) or a small hairpin RNA(“shRNA”). Methods for constructing interfering RNAs are well known inthe art. For example, the interfering RNA can be assembled from twoseparate oligonucleotides, where one strand is the sense strand and theother is the antisense strand, wherein the antisense and sense strandsare self-complementary (i.e., each strand comprises nucleotide sequencethat is complementary to nucleotide sequence in the other strand; suchas where the antisense strand and sense strand form a duplex or doublestranded structure); the antisense strand comprises nucleotide sequencethat is complementary to a nucleotide sequence in a target nucleic acidmolecule or a portion thereof (i.e., an undesired gene) and the sensestrand comprises nucleotide sequence corresponding to the target nucleicacid sequence or a portion thereof. Alternatively, interfering RNA isassembled from a single oligonucleotide, where the self-complementarysense and antisense regions are linked by means of nucleic acid based ornon-nucleic acid-based linker(s). The interfering RNA can be apolynucleotide with a duplex, asymmetric duplex, hairpin or asymmetrichairpin secondary structure, having self-complementary sense andantisense regions, wherein the antisense region comprises a nucleotidesequence that is complementary to nucleotide sequence in a separatetarget nucleic acid molecule or a portion thereof and the sense regionhaving nucleotide sequence corresponding to the target nucleic acidsequence or a portion thereof. The interfering can be a circularsingle-stranded polynucleotide having two or more loop structures and astem comprising self-complementary sense and antisense regions, whereinthe antisense region comprises nucleotide sequence that is complementaryto nucleotide sequence in a target nucleic acid molecule or a portionthereof and the sense region having nucleotide sequence corresponding tothe target nucleic acid sequence or a portion thereof, and wherein thecircular polynucleotide can be processed either in vivo or in vitro togenerate an active siRNA molecule capable of mediating RNA interference.

In some embodiments, the interfering RNA coding region encodes aself-complementary RNA molecule having a sense region, an antisenseregion and a loop region. Such an RNA molecule when expressed desirablyforms a “hairpin” structure, and is referred to herein as a “shRNA.” Theloop region is generally between about 2 and about 10 nucleotides inlength. In some embodiments, the loop region is from about 6 to about 9nucleotides in length. In some embodiments, the sense region and theantisense region are between about 15 and about 20 nucleotides inlength. Following post-transcriptional processing, the small hairpin RNAis converted into a siRNA by a cleavage event mediated by the enzymeDicer, which is a member of the RNase III family. The siRNA is thencapable of inhibiting the expression of a gene with which it shareshomology. For details, see Brummelkamp et al., Science 296:550-553,(2002); Lee et al, Nature Biotechnol., 20, 500-505, (2002); Miyagishiand Taira, Nature Biotechnol 20:497-500, (2002); Paddison et al. Genes &Dev. 16:948-958, (2002); Paul, Nature Biotechnol, 20, 505-508, (2002);Sui, Proc. Natl. Acad. Sd. USA, 99(6), 5515-5520, (2002); Yu et al. ProcNatl Acad Sci USA 99:6047-6052, (2002).

The target RNA cleavage reaction guided by siRNAs is highly sequencespecific. In general, siRNA containing a nucleotide sequences identicalto a portion of the target nucleic acid are preferred for inhibition.However, 100% sequence identity between the siRNA and the target gene isnot required to practice the present invention. Thus the invention hasthe advantage of being able to tolerate sequence variations that mightbe expected due to genetic mutation, strain polymorphism, orevolutionary divergence. For example, siRNA sequences with insertions,deletions, and single point mutations relative to the target sequencehave also been found to be effective for inhibition. Alternatively,siRNA sequences with nucleotide analog substitutions or insertions canbe effective for inhibition. In general the siRNAs must retainspecificity for their target, i.e., must not directly bind to, ordirectly significantly affect expression levels of, transcripts otherthan the intended target.

Ribozymes

In some embodiments, the inhibitory nucleic acids are ribozymes.Trans-cleaving enzymatic nucleic acid molecules can also be used; theyhave shown promise as therapeutic agents for human disease (Usman &McSwiggen, 1995 Ann. Rep. Med. Chem. 30, 285-294; Christoffersen andMarr, 1995 J. Med. Chem. 38, 2023-2037). Enzymatic nucleic acidmolecules can be designed to cleave specific RNA targets within thebackground of cellular RNA. Such a cleavage event renders the RNAnon-functional.

In general, enzymatic nucleic acids with RNA cleaving activity act byfirst binding to a target RNA. Such binding occurs through the targetbinding portion of an enzymatic nucleic acid which is held in closeproximity to an enzymatic portion of the molecule that acts to cleavethe target RNA. Thus, the enzymatic nucleic acid first recognizes andthen binds a target RNA through complementary base pairing, and oncebound to the correct site, acts enzymatically to cut the target RNA.Strategic cleavage of such a target RNA will destroy its ability todirect synthesis of an encoded protein. After an enzymatic nucleic acidhas bound and cleaved its RNA target, it is released from that RNA tosearch for another target and can repeatedly bind and cleave newtargets.

Several approaches such as in vitro selection (evolution) strategies(Orgel, 1979, Proc. R. Soc. London, B 205, 435) have been used to evolvenew nucleic acid catalysts capable of catalyzing a variety of reactions,such as cleavage and ligation of phosphodiester linkages and amidelinkages, (Joyce, 1989, Gene, 82, 83-87; Beaudry et al., 1992, Science257, 635-641; Joyce, 1992, Scientific American 267, 90-97; Breaker etal, 1994, TIBTECH 12, 268; Bartel et al, 1993, Science 261 :1411-1418;Szostak, 1993, TIBS 17, 89-93; Kumar et al, 1995, FASEB J., 9, 1183;Breaker, 1996, Curr. Op. Biotech., 1, 442). The development of ribozymesthat are optimal for catalytic activity would contribute significantlyto any strategy that employs RNA-cleaving ribozymes for the purpose ofregulating gene expression. The hammerhead ribozyme, for example,functions with a catalytic rate (kcat) of about 1 min ⁻¹ in the presenceof saturating (10 mM) concentrations of Mg²⁺ cofactor. An artificial“RNA ligase” ribozyme has been shown to catalyze the correspondingself-modification reaction with a rate of about 100 min⁻¹. In addition,it is known that certain modified hammerhead ribozymes that havesubstrate binding arms made of DNA catalyze RNA cleavage with multipleturn-over rates that approach 100 min⁻¹.

Making and Using Inhibitory Nucleic Acids

The nucleic acid sequences used to practice the methods describedherein, whether RNA, cDNA, genomic DNA, vectors, viruses or hybridsthereof, can be isolated from a variety of sources, geneticallyengineered, amplified, and/or expressed/generated recombinantly. Ifdesired, nucleic acid sequences of the invention can be inserted intodelivery vectors and expressed from transcription units within thevectors. The recombinant vectors can be DNA plasmids or viral vectors.Generation of the vector construct can be accomplished using anysuitable genetic engineering techniques well known in the art,including, without limitation, the standard techniques of PCR,oligonucleotide synthesis, restriction endonuclease digestion, ligation,transformation, plasmid purification, and DNA sequencing, for example asdescribed in Sambrook et al. Molecular Cloning: A Laboratory Manual.(1989)), Coffin et al. (Retroviruses. (1997)) and “RNA Viruses: APractical Approach” (Alan J. Cann, Ed., Oxford University Press,(2000)).

Preferably, inhibitory nucleic acids of the invention are synthesizedchemically. Nucleic acid sequences used to practice this invention canbe synthesized in vitro by well-known chemical synthesis techniques, asdescribed in, e.g., Adams (1983) J. Am. Chem. Soc. 105:661; Belousov(1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol.Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang(1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109;Beaucage (1981) Tetra. Lett. 22:1859; U.S. Pat. No. 4,458,066;WO/2008/043753 and WO/2008/049085, and the references cited therein.

Nucleic acid sequences of the invention can be stabilized againstnucleolytic degradation such as by the incorporation of a modification,e.g., a nucleotide modification. For example, nucleic acid sequences ofthe invention includes a phosphorothioate at least the first, second, orthird internucleotide linkage at the 5′ or 3′ end of the nucleotidesequence. As another example, the nucleic acid sequence can include a2′-modified nucleotide, e.g., a 2′-deoxy, 2′-deoxy-2′-fluoro,2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl (2′-O-AP),2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl(2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or2′-O—N-methylacetamido (2′-O—NMA). As another example, the nucleic acidsequence can include at least one 2′-O-methyl-modified nucleotide, andin some embodiments, all of the nucleotides include a 2′-O-methylmodification. In some embodiments, the nucleic acids are “locked,” i.e.,comprise nucleic acid analogues in which the ribose ring is “locked” bya methylene bridge connecting the 2′-O atom and the 4′-C atom (see,e.g., Kaupinnen et al., Drug Disc. Today 2(3):287-290 (2005); Koshkin etal., J. Am. Chem. Soc., 120(50):13252-13253 (1998)). For additionalmodifications see US 20100004320, US 20090298916, and US 20090143326.

It is understood that any of the modified chemistries or formats ofinhibitory nucleic acids described herein can be combined with eachother, and that one, two, three, four, five, or more different types ofmodifications can be included within the same molecule.

Techniques for the manipulation of nucleic acids used to practice thisinvention, such as, e.g., subcloning, labeling probes (e.g.,random-primer labeling using Klenow polymerase, nick translation,amplification), sequencing, hybridization and the like are welldescribed in the scientific and patent literature, see, e.g., Sambrooket al., Molecular Cloning; A Laboratory Manual 3d ed. (2001); CurrentProtocols in Molecular Biology, Ausubel et al., eds. (John Wiley & Sons,Inc., New York 2010); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); Laboratory Techniques In Biochemistry AndMolecular Biology: Hybridization With Nucleic Acid Probes, Part I.Theory and Nucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993).

Pharmaceutical Compositions

The methods described herein can include the administration ofpharmaceutical compositions and formulations comprising inhibitorynucleic acid sequences designed to target an RNA.

In some embodiments, the compositions are formulated with apharmaceutically acceptable carrier. The pharmaceutical compositions andformulations can be administered parenterally, topically, orally or bylocal administration, such as by aerosol or transdermally. Thepharmaceutical compositions can be formulated in any way and can beadministered in a variety of unit dosage forms depending upon thecondition or disease and the degree of illness, the general medicalcondition of each patient, the resulting preferred method ofadministration and the like. Details on techniques for formulation andadministration of pharmaceuticals are well described in the scientificand patent literature, see, e.g., Remington: The Science and Practice ofPharmacy, 21st ed., 2005.

The inhibitory nucleic acids can be administered alone or as a componentof a pharmaceutical formulation (composition). The compounds may beformulated for administration, in any convenient way for use in human orveterinary medicine. Wetting agents, emulsifiers and lubricants, such assodium lauryl sulfate and magnesium stearate, as well as coloringagents, release agents, coating agents, sweetening, flavoring andperfuming agents, preservatives and antioxidants can also be present inthe compositions.

Formulations of the compositions of the invention include those suitablefor intradermal, inhalation, oral/nasal, topical, parenteral, rectal,and/or intravaginal administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient (e.g.,nucleic acid sequences of this invention) which can be combined with acarrier material to produce a single dosage form will vary dependingupon the host being treated, the particular mode of administration,e.g., intradermal or inhalation. The amount of active ingredient whichcan be combined with a carrier material to produce a single dosage formwill generally be that amount of the compound which produces atherapeutic effect, e.g., an antigen specific T cell or humoralresponse.

Pharmaceutical formulations of this invention can be prepared accordingto any method known to the art for the manufacture of pharmaceuticals.Such drugs can contain sweetening agents, flavoring agents, coloringagents and preserving agents. A formulation can be admixtured withnontoxic pharmaceutically acceptable excipients which are suitable formanufacture. Formulations may comprise one or more diluents,emulsifiers, preservatives, buffers, excipients, etc. and may beprovided in such forms as liquids, powders, emulsions, lyophilizedpowders, sprays, creams, lotions, controlled release formulations,tablets, pills, gels, on patches, in implants, etc.

Pharmaceutical formulations for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art inappropriate and suitable dosages. Such carriers enable thepharmaceuticals to be formulated in unit dosage forms as tablets, pills,powder, dragees, capsules, liquids, lozenges, gels, syrups, slurries,suspensions, etc., suitable for ingestion by the patient. Pharmaceuticalpreparations for oral use can be formulated as a solid excipient,optionally grinding a resulting mixture, and processing the mixture ofgranules, after adding suitable additional compounds, if desired, toobtain tablets or dragee cores. Suitable solid excipients arecarbohydrate or protein fillers include, e.g., sugars, includinglactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice,potato, or other plants; cellulose such as methyl cellulose,hydroxypropylmethyl-cellulose, or sodium carboxy-methylcellulose; andgums including arabic and tragacanth; and proteins, e.g., gelatin andcollagen. Disintegrating or solubilizing agents may be added, such asthe cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a saltthereof, such as sodium alginate. Push-fit capsules can contain activeagents mixed with a filler or binders such as lactose or starches,lubricants such as talc or magnesium stearate, and, optionally,stabilizers. In soft capsules, the active agents can be dissolved orsuspended in suitable liquids, such as fatty oils, liquid paraffin, orliquid polyethylene glycol with or without stabilizers.

Aqueous suspensions can contain an active agent (e.g., nucleic acidsequences of the invention) in admixture with excipients suitable forthe manufacture of aqueous suspensions, e.g., for aqueous intradermalinjections. Such excipients include a suspending agent, such as sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia,and dispersing or wetting agents such as a naturally occurringphosphatide (e.g., lecithin), a condensation product of an alkyleneoxide with a fatty acid (e.g., polyoxyethylene stearate), a condensationproduct of ethylene oxide with a long chain aliphatic alcohol (e.g.,heptadecaethylene oxycetanol), a condensation product of ethylene oxidewith a partial ester derived from a fatty acid and a hexitol (e.g.,polyoxyethylene sorbitol mono-oleate), or a condensation product ofethylene oxide with a partial ester derived from fatty acid and ahexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate). Theaqueous suspension can also contain one or more preservatives such asethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one ormore flavoring agents and one or more sweetening agents, such assucrose, aspartame or saccharin. Formulations can be adjusted forosmolarity.

In some embodiments, oil-based pharmaceuticals are used foradministration of nucleic acid sequences of the invention. Oil-basedsuspensions can be formulated by suspending an active agent in avegetable oil, such as arachis oil, olive oil, sesame oil or coconutoil, or in a mineral oil such as liquid paraffin; or a mixture of these.See e.g., U.S. Pat. No. 5,716,928 describing using essential oils oressential oil components for increasing bioavailability and reducinginter- and intra-individual variability of orally administeredhydrophobic pharmaceutical compounds (see also U.S. Pat. No. 5,858,401).The oil suspensions can contain a thickening agent, such as beeswax,hard paraffin or cetyl alcohol. Sweetening agents can be added toprovide a palatable oral preparation, such as glycerol, sorbitol orsucrose. These formulations can be preserved by the addition of anantioxidant such as ascorbic acid. As an example of an injectable oilvehicle, see Minto (1997) J. Pharmacol. Exp. Ther. 281:93-102.

Pharmaceutical formulations can also be in the form of oil-in-wateremulsions. The oily phase can be a vegetable oil or a mineral oil,described above, or a mixture of these. Suitable emulsifying agentsinclude naturally-occurring gums, such as gum acacia and gum tragacanth,naturally occurring phosphatides, such as soybean lecithin, esters orpartial esters derived from fatty acids and hexitol anhydrides, such assorbitan mono-oleate, and condensation products of these partial esterswith ethylene oxide, such as polyoxyethylene sorbitan mono-oleate. Theemulsion can also contain sweetening agents and flavoring agents, as inthe formulation of syrups and elixirs. Such formulations can alsocontain a demulcent, a preservative, or a coloring agent. In alternativeembodiments, these injectable oil-in-water emulsions of the inventioncomprise a paraffin oil, a sorbitan monooleate, an ethoxylated sorbitanmonooleate and/or an ethoxylated sorbitan trioleate.

The pharmaceutical compounds can also be administered by in intranasal,intraocular and intravaginal routes including suppositories,insufflation, powders and aerosol formulations (for examples of steroidinhalants, see e.g., Rohatagi (1995) J. Clin. Pharmacol. 35:1187-1193;Tjwa (1995) Ann. Allergy Asthma Immunol. 75:107-111). Suppositoriesformulations can be prepared by mixing the drug with a suitablenon-irritating excipient which is solid at ordinary temperatures butliquid at body temperatures and will therefore melt in the body torelease the drug. Such materials are cocoa butter and polyethyleneglycols.

In some embodiments, the pharmaceutical compounds can be deliveredtransdermally, by a topical route, formulated as applicator sticks,solutions, suspensions, emulsions, gels, creams, ointments, pastes,jellies, paints, powders, and aerosols.

In some embodiments, the pharmaceutical compounds can also be deliveredas microspheres for slow release in the body. For example, microspherescan be administered via intradermal injection of drug which slowlyrelease subcutaneously; see Rao (1995) J. Biomater Sci. Polym. Ed.7:623-645; as biodegradable and injectable gel formulations, see, e.g.,Gao (1995) Pharm. Res. 12:857-863 (1995); or, as microspheres for oraladministration, see, e.g., Eyles (1997) J. Pharm. Pharmacol. 49:669-674.

In some embodiments, the pharmaceutical compounds can be parenterallyadministered, such as by intravenous (IV) administration oradministration into a body cavity or lumen of an organ. Theseformulations can comprise a solution of active agent dissolved in apharmaceutically acceptable carrier. Acceptable vehicles and solventsthat can be employed are water and Ringer's solution, an isotonic sodiumchloride. In addition, sterile fixed oils can be employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid can likewise be used in the preparation ofinjectables. These solutions are sterile and generally free ofundesirable matter. These formulations may be sterilized byconventional, well known sterilization techniques. The formulations maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjusting agents, e.g., sodium acetate, sodiumchloride, potassium chloride, calcium chloride, sodium lactate and thelike. The concentration of active agent in these formulations can varywidely, and will be selected primarily based on fluid volumes,viscosities, body weight, and the like, in accordance with theparticular mode of administration selected and the patient's needs. ForIV administration, the formulation can be a sterile injectablepreparation, such as a sterile injectable aqueous or oleaginoussuspension. This suspension can be formulated using those suitabledispersing or wetting agents and suspending agents. The sterileinjectable preparation can also be a suspension in a nontoxicparenterally-acceptable diluent or solvent, such as a solution of1,3-butanediol. The administration can be by bolus or continuousinfusion (e.g., substantially uninterrupted introduction into a bloodvessel for a specified period of time).

In some embodiments, the pharmaceutical compounds and formulations canbe lyophilized. Stable lyophilized formulations comprising an inhibitorynucleic acid can be made by lyophilizing a solution comprising apharmaceutical of the invention and a bulking agent, e.g., mannitol,trehalose, raffinose, and sucrose or mixtures thereof. A process forpreparing a stable lyophilized formulation can include lyophilizing asolution about 2.5 mg/mL protein, about 15 mg/mL sucrose, about 19 mg/mLNaCl, and a sodium citrate buffer having a pH greater than 5.5 but lessthan 6.5. See, e.g., U.S. 20040028670.

The compositions and formulations can be delivered by the use ofliposomes. By using liposomes, particularly where the liposome surfacecarries ligands specific for target cells, or are otherwisepreferentially directed to a specific organ, one can focus the deliveryof the active agent into target cells in vivo. See, e.g., U.S. Pat. Nos.6,063,400; and 6,007,839; Al-Muhammed (1996) J. Microencapsul.13:293-306; Chonn (1995) Curr. Opin. Biotechnol. 6:698-708; Ostro (1989)Am. J. Hosp. Pharm. 46:1576-1587. As used in the present invention, theterm “liposome” means a vesicle composed of amphiphilic lipids arrangedin a bilayer or bilayers. Liposomes are unilamellar or multilamellarvesicles that have a membrane formed from a lipophilic material and anaqueous interior that contains the composition to be delivered. Cationicliposomes are positively charged liposomes that are believed to interactwith negatively charged DNA molecules to form a stable complex.Liposomes that are pH-sensitive or negatively-charged are believed toentrap DNA rather than complex with it. Both cationic and noncationicliposomes have been used to deliver DNA to cells.

Liposomes can also include “sterically stabilized” liposomes, i.e.,liposomes comprising one or more specialized lipids. When incorporatedinto liposomes, these specialized lipids result in liposomes withenhanced circulation lifetimes relative to liposomes lacking suchspecialized lipids. Examples of sterically stabilized liposomes arethose in which part of the vesicle-forming lipid portion of the liposomecomprises one or more glycolipids or is derivatized with one or morehydrophilic polymers, such as a polyethylene glycol (PEG) moiety.Liposomes and their uses are further described in U.S. Pat. No.6,287,860.

The formulations of the invention can be administered for prophylacticand/or therapeutic treatments. In some embodiments, for therapeuticapplications, compositions are administered to a subject who is need ofreduced triglyceride levels, or who is at risk of or has a disorderdescribed herein, in an amount sufficient to cure, alleviate orpartially arrest the clinical manifestations of the disorder or itscomplications; this can be called a therapeutically effective amount.For example, in some embodiments, pharmaceutical compositions of theinvention are administered in an amount sufficient to decrease serumlevels of triglycerides in the subject.

The amount of pharmaceutical composition adequate to accomplish this isa therapeutically effective dose. The dosage schedule and amountseffective for this use, i.e., the dosing regimen, will depend upon avariety of factors, including the stage of the disease or condition, theseverity of the disease or condition, the general state of the patient'shealth, the patient's physical status, age and the like. In calculatingthe dosage regimen for a patient, the mode of administration also istaken into consideration.

The dosage regimen also takes into consideration pharmacokineticsparameters well known in the art, i.e., the active agents' rate ofabsorption, bioavailability, metabolism, clearance, and the like (see,e.g., Hidalgo-Aragones (1996) J. Steroid Biochem. Mol. Biol. 58:611-617;Groning (1996) Pharmazie 51:337-341; Fotherby (1996) Contraception54:59-69; Johnson (1995) J. Pharm. Sci. 84:1144-1146; Rohatagi (1995)Pharmazie 50:610-613; Brophy (1983) Eur. J. Clin. Pharmacol. 24:103-108;Remington: The Science and Practice of Pharmacy, 21st ed., 2005). Thestate of the art allows the clinician to determine the dosage regimenfor each individual patient, active agent and disease or conditiontreated. Guidelines provided for similar compositions used aspharmaceuticals can be used as guidance to determine the dosageregiment, i.e., dose schedule and dosage levels, administered practicingthe methods of the invention are correct and appropriate.

Single or multiple administrations of formulations can be givendepending on for example: the dosage and frequency as required andtolerated by the patient, the degree and amount of therapeutic effectgenerated after each administration (e.g., effect on tumor size orgrowth), and the like. The formulations should provide a sufficientquantity of active agent to effectively treat, prevent or ameliorateconditions, diseases or symptoms.

In alternative embodiments, pharmaceutical formulations for oraladministration are in a daily amount of between about 1 to 100 or moremg per kilogram of body weight per day. Lower dosages can be used, incontrast to administration orally, into the blood stream, into a bodycavity or into a lumen of an organ. Substantially higher dosages can beused in topical or oral administration or administering by powders,spray or inhalation. Actual methods for preparing parenterally ornon-parenterally administrable formulations will be known or apparent tothose skilled in the art and are described in more detail in suchpublications as Remington: The Science and Practice of Pharmacy, 21sted., 2005.

Various studies have reported successful mammalian dosing usingcomplementary nucleic acid sequences. For example, Esau C., et al.,(2006) Cell

Metabolism, 3(2):87-98 reported dosing of normal mice withintraperitoneal doses of miR-122 antisense oligonucleotide ranging from12.5 to 75 mg/kg twice weekly for 4 weeks. The mice appeared healthy andnormal at the end of treatment, with no loss of body weight or reducedfood intake. Plasma transaminase levels were in the normal range (AST3/4 45, ALT 3/4 35) for all doses with the exception of the 75 mg/kgdose of miR-122 ASO, which showed a very mild increase in ALT and ASTlevels. They concluded that 50 mg/kg was an effective, non-toxic dose.Another study by Krützfeldt J., et al., (2005) Nature 438, 685-689,injected anatgomirs to silence miR-122 in mice using a total dose of 80,160 or 240 mg per kg body weight. The highest dose resulted in acomplete loss of miR-122 signal. In yet another study, locked nucleicacid molecules (“LNA molecules”) were successfully applied in primatesto silence miR-122. Elmen J., et al., (2008) Nature 452, 896-899, reportthat efficient silencing of miR-122 was achieved in primates by threedoses of 10 mg kg-1 LNA-antimiR, leading to a long-lasting andreversible decrease in total plasma cholesterol without any evidence forLNA-associated toxicities or histopathological changes in the studyanimals.

In some embodiments, the methods described herein can includeco-administration with other drugs or pharmaceuticals, e.g.,compositions for providing cholesterol homeostasis. For example, theinhibitory nucleic acids can be co-administered with other treatmentoptions for treating or reducing risk of PDAC, e.g., surgery (e.g., aWhipple's operation), chemotherapy (e.g., gemcitabine (GEMZAR®),FOLFIRINOX, Nab-paclitaxel (ABRAXANE®), fluorouracil (5-FU),capecitabine (XELODA®), oxaliplatin (ELOXATIN®)), radiotherapy, and/orirreversible electroporation (e.g., NANOKNIFE®).

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Experimental Procedures

The following methods were used in the Examples set forth below, unlessotherwise noted.

Mice

Mice were housed in pathogen-free animal facilities. All experimentswere conducted under protocol 2007N000200 approved by the Subcommitteeon Research Animal Care at Massachusetts General Hospital. Mice weremaintained on a mixed 129SV/C57BL/6 background. Data presented includeboth male and female mice. All mice included in the survival analysiswere euthanized when criteria for disease burden were reached.

Sirt6^(flox/flox) conditional strain (Sebastian et al., 2012) werecrossed with the p48-Cre strain (Kawaguchi et al., 2002), theconditional p53^(flox) strain (Marino et al., 2000) and theLSL-Kras^(G12D) strain (Jackson et al., 2001) which consists of a mutantKras^(G12D) allele knocked into the endogenous Kras locus, preceded byan LSL cassette.

Cell Lines

Pancreatic cancer cell lines, HPAFII, KP4, MiaPaCa, SW1990, YAPC, 8988T,PSN1, 89a, Panc03.27, Panc-1, BxPc3, HPAC, Panc8.13, ASPC-1, Su86.86 andHPNE were obtained from American Type Culture Collection (ATCC;Rockville, Md.), and grown in their required growth medium per ATCCdescription. The DanG pancreatic cancer cells were obtained from DSMZ,SUIT-2 were obtained from JCRB cell bank and COLO357 was a gift fromPaul Chiao (MD Anderson). All were grown in RPMI 1640 supplemented with10% fetal bovine serum and 1% penicillin (100 U/ml)/streptomycin (100Ug/ml) (Invitrogen Gibco). Human pancreatic ductal epithelial cells(HPDE) (Furukawa et al., 1996) were obtained from Ming Tsao (Universityof Toronto) and grown in keratinocyte serum-free (KSF) medium with 0.2ng/ml EGF and 30 μg/ml bovine pituitary extract (Invitrogen Gibco,Carlsbad, Calif. ) at 37° C. under 5% CO2. To establish mouse pancreaticcancer cell lines, freshly isolated tumor specimens from Sirt6^(ff);Kras^(G12D); p53^(f/+); p48-Cre (SIRT6 KO) and Sirt6^(+/+); Kras^(G12D);p53^(f/+); p48-Cre (SIRT6 WT) mice were minced with sterile razorblades, digested with trypsin for 30 minutes at 37° C., and thenresuspended in RPMI 1640 and supplemented with 10% fetal bovine serumand 1% penicillin (100 U/ml)/streptomycin (100 Ug/ml) (Invitrogen Gibco)and seeded on plates coated with rat tail collagen (BD Biosciences).Cells were passaged by trypsinization. All studies were done on cellscultivated for less than ten passages. SIRT6 knockout (KO) primary mouseembryonic fibroblast (MEFs) were generated from 13.5-day-old embryos asdescribed (Mostoslaysky et al., 2006). These cells were immortalized byusing the standard 3T3 protocol. Cells were cultured in high glucoseDulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetalbovine serum, 1% penicillin (100 U/ml)/streptomycin (100 Ug/ml)(Invitrogen), 2 mM L-glutamine, 0.1mM NEAA, 1mM sodium pyruvate, and 20mM HEPES.

Constructs and Viral Infection

Full-length wild-type SIRT6 cDNA (variant 1; NM_016539.2) was amplifiedfrom the HPDE cells using the following primers(CAGGATCCTTGTTCCCGTGGGGCAGTCGAGG (SEQ ID NO:1); bold sequence indicatesBamHI site) and (CAGAATTCCTACAAAAAGCCCCACCCTCCC (SEQ ID NO:2); boldsequence indicates EcoRI site). Following PCR amplification andsubcloning into pGEMT (Promega), SIRT6 constructs were digested withBamHI and EcoRI, and purified with the QIAquick Gel extraction kit(Qiagen). Digested SIRT6 was subcloned into pRetroX-TIGHT-Pur plasmid(Clontech) and site-directed mutagenesis of wild-type SIRT6 used theQuikChange Lightning site-directed mutagenesis kit (Stratagene, LaJolla, Calif., USA) to generate the H133Y, catalytic dead mutant.pLVX-Tet-On was obtained from Clontech. pMSCV-3xFlag-SIRT6 waspreviously described in (Sebastian et al., 2012).

The following lentiviral plasmids were used: Human pTRIPz-shSIRT6(Dharmacon RHS4740) and negative control shRNA vector was a kind giftfrom David Lombard. Human and mouse (target sequence conserved)pLKO.1-shLIN28B vector (TRCN0000122599) target sequence:5′-GCCTTGAGTCAATACGGGTAA-3′ (SEQ ID NO:3); pLKO.1-shHMGA2 vector(TRCN0000021965) target sequence: 5′-GCCACAACAAGTTGTTCAGAA-3′ (SEQ IDNO:4); pLKO.1-shMYC-2 vector (TRCN0000039639) target sequence:5′-CCCAAGGTAGTTATCCTTAAA-3′ (SEQ ID NO:5); pLKO.1-shMYC-5 vector(TRCN0000039642) target sequence: 5′-CCTGAGACAGATCAGCAACAA-3′ (SEQ IDNO:6); pLKO.1-shIGF2BP3-1 vector (TRCN0000074673) target sequence:5′-GCCTCATTCTTATTTCAAGAT-3′ (SEQ ID NO:7); pLKO.1-shIGF2BP3-3 vector(TRCN0000074675) target sequence: 5′ -CGGTGAATGAACTTCAGAATT-3′ (SEQ IDNO:8); pLKO.1-shIGF2BP1-2 vector (TRCN0000075149) target sequence:5′-GCAGTGGTGAATGTCACCTAT-3′ (SEQ ID NO:9); pLKO.1-shIGF2BP1-5 vector(TRCN0000075152) target sequence: 5′-CCTGGCCCATAATAACTTTGT-3′ (SEQ IDNO:10); mouse pLKO.1-shPdk1 vector (TRCN0000078808) target sequence:5′-GCTGAGTATTTCTTTCAAGTT-3′ (SEQ ID NO:11) and mouse pLKO1-shLdha-2vector (TRCN0000041744) target sequence: 5′-CGTGAACATCTTCAAGTTCAT-3′(SEQ ID NO:12) and mouse pLKO.1 shLdha-5 vector (TRCN0000041747) targetsequence: 5′-CGTCTCCCTGAAGTCTCTTAA-3′ (SEQ ID NO:13) were obtained fromthe MGH RNAi Consortium Library. Mouse pLKO.1-shMYC vector(TRCN0000042514) target sequence: 5′-GCCTACATCCTGTCCATTCAA-3′ (SEQ IDNO:14; Open Biosciences). pLKO.1 shRNA (shCtl) with target sequence5′-GCAAGCTGACCCTGAAGTTCAT-3′ (SEQ ID NO:15) was used as negative controlshRNA. pBabe let-7g 7S21L mutant construct was a kind gift from RichardGregory.

Viral particles containing the above mentioned plasmids were synthesizedusing either lentiviral (pCMV-dR8.91) or retroviral (pCL-ECO) packagingplasmids with pCMV-VSV-G (Addgene). Cells were infected by incubatingwith virus and 10 μg/ml polybrene. Twenty-four hours later, cells wereselected in 2.5 μg/ml puromycin for at least two days and the pooledpopulations were used for various experiments. For all experimentsinvolving the dox-inducible pRetro construct cells were treated with 1μg/mL dox for 48 hours and for the dox-inducible shSIRT6 cells weretreated with 1 μg/mL dox for 72 hours unless otherwise indicated.

Silencer Select siRNA were purchased from Ambion and 10 nM wastransfected into cells with Lipofectamine RNAiMax (Invitrogen). Negativecontrol #1 (4390843), mouse siLin28b #1 (4390771 s117291), mousesiLin28b #2 (4390771 s117292), mouse siIgf2bp3 (4390771 s100444) humansiLin28b #1 (4392420 s52477) and human siLin28b #2 (4392420 s52478).miRCURY LNA Let-7 mimetics were purchased from Exiquon and 50 nM wasreverse transfected into cells with Lipofectamine RNAiMax (Invitrogen).Negative control mimetic (479903-001), hsa-let7c-5p (471696-001) andhsa-let7d-5p (470030-001).

Gel Electrophoresis and Western Blotting

Chromatin fractions were prepared by resuspending the cell pellet inlysis buffer containing 10mM HEPES pH 7.4, 10 mM KCl, 0.05% NP-40supplemented with a protease inhibitor cocktail (Complete EDTA-free,Roche Applied Science), 5 μM TSA, 5 mM sodium butyrate, 1 mM DTT, 1 mMPMSF, 50 mM NaF, 0.2 mM sodium orthovanadate and phosphatase inhibitors(Phosphatase Inhibitor Cocktail Sets I and II, Calbiochem) and incubatedon ice for 20 minutes. The lysate was then centrifuged at 14,000 rpm for10 minutes at 4° C. The supernatant was removed (cytosolic fraction) andthe pellet (nuclei) was acid-extracted using 0.2N HCl and incubated onice for 20 minutes. The lysate was then centrifuged at 14,000 rpm for 10minutes at 4° C. The supernatant (contains acid soluble proteins) wasneutralized using 1M Tris-HCl pH 8. For whole cell lysate (WCL), thecell pellet was resuspended in RIPA buffer supplemented with a proteaseinhibitor cocktail (Complete EDTA-free, Roche Applied Science), 5 μMTSA, 5 mM sodium butyrate, 1 mM DTT, 1 mM PMSF, 50 mM NaF, 0.2 mM sodiumorthovanadate and phosphatase inhibitors (Phosphatase Inhibitor CocktailSets I and II, Calbiochem) and incubated on ice for 20 minutes. Thelysate was then centrifuged at 14,000 rpm for 10 minutes at 4° C. andthe supernatant was harvested. Protein concentration was quantified byBiorad Protein Assay. Ten-micrograms protein (chromatin) 20 μg (WCL) waselectrophoresed on a 10-20% gradient polyacrylamide gel with SDS(Biorad) and electroblotted onto polyvinylidene difluoride membranes(PVDF) (Millipore). Membranes were blocked in TBS with 5% non-fat milkand 0.1% Tween and probed with anti-SIRT6 (Abcam, ab62739), anti-H3K9Ac(Millipore 07-352), anti-H3K56Ac (ab76307), anti-Lin28b (ab71415 andCell Signaling 4196), anti-PDK1 (Cell signaling #3820), anti-Myc(ab32072), anti-IGF2BP1 (Cell Signaling #8482), anti-HMGA2 (CellSignaling #8179), anti-IGF2BP3 (Proteintech 14642-1-AP) and anti-β actin(Sigma A5316) or total-H3 (ab1791) as a loading control. Bound proteinswere detected with horseradish-peroxidase-conjugated secondaryantibodies (Vector Biolaboratories) and SuperSignal West PicoLuminol/Enhancer Solution (Thermo Scientific).

Glucose Uptake Assay

Cells were grown under normal conditions for 24 hours and 100 μM 2-NBDG(Invitrogen) was added to the media for 2 hours. Fluorescence wasmeasured by flow cytometry using a FACSCalibur Analyzer (BD). Data areshown as mean±std between duplicates and are representative of twoindependent experiments.

Real-Time RT-PCR Analysis

Total RNA was extracted with the TriPure Isolation Reagent (Roche) asdescribed by the manufacturer. For cDNA synthesis, 1 μg of total RNA wasreverse-transcribed by using the QuantiTect Reverse Transcription Kit(Qiagen). Real-time PCR was run in duplicate using SYBR green master mix(Roche), following the manufacturer's instructions, with the exceptionthat the final volume was 12.5 μl of SYBR green reaction mix. Real-timemonitoring of PCR amplification was performed using the LightCycler 480detection system (Roche). Data were expressed as relative mRNA levelsnormalized to the β-actin expression level in each sample and arerepresented as mean±s.e.m. between two independent experiments unlessotherwise indicated in the figure legend.

MicroRNA Sequencing Analysis

Next-generation sequencing of small RNA-Seq for PLKO and shLIN28bknockdown samples (three replicates each) was performed using IlluminaHiSeq instrument, resulting in approximately 35 million pairs of 50-bpreads per sample. These reads were aligned and miRNA expression profileswere generated using miRExpress (Wang et al., 2009), followed by theanalysis of differential expression using edgeR package (Robinson etal., 2010).

Tumor Sphere Assay

Cells were plated as single-cell suspension in ultralow attachment24-well plates (Corning) and grown in DMEM/F12 medium (serum free)supplemented with 20 μl ml⁻¹ B27 (Invitrogen), 20 ng ml⁻¹ EGF and 20 ngml⁻¹ bFGF. Fresh media (300 μl) was added every 3 days. Tumor sphereswere counted and photographed at day 10. Tumor sphere assay wasperformed in triplicate, and are represented as mean±s.e.m. betweenthree independent experiments.

Xenografts

For murine PDAC xenografts 2X10⁴ cells were injected subcutaneously intothe flanks of SCID mice (Charles River). For human PDAC xenografts 5X10⁶cells in 100 μl PBS: 100 μl Matrigel (Corning) was injectedsubcutaneously into the flanks of SCID mice (Charles River) For bothmodels, mice were checked for the appearance of tumors twice per week,and the tumors were harvested when they reached ˜100 mm in diameter.

Histology and Immunostaining

Pancreata were harvested, submitted for histological examination, andanalyzed in a blinded fashion by pathologist (V.D.). For quantificationof PanIN and PDAC, a grading scheme endorsed by the WHO (Aaltonen etal., 2000) we used, which is based primarily on the extent of glandformation. Tissue samples were fixed overnight in 4% bufferedformaldehyde, and then embedded in paraffin and sectioned (5 μmthickness) by the DF/HCC Research Pathology Core. Haematoxylin and eosinstaining was performed using standard methods. Tissue microarrays wereconstructed from formalin-fixed paraffin embedded tissue with each coremeasuring 3 mm in diameter. Immunohistochemistry was performed aspreviously described (Fitamant et al., 2015). Primary antibodies werediluted in blocking solution as follows: anti-Lin28b (LS Bio LS-B3423)1:200 for mouse and human tissues; anti-SIRT6 (Cell SignalingTechnology, #12486) 1:300 for mouse tissues and 1:200 for human tissues.Stained slides were photographed with an Olympus DP72 microscope.Immunohistochemistry was scored semi quantitatively, in a blindedfashion by pathologist (V.D.) on a 0 (no staining) to 3 (strongestintensity) scale based on the intensity of reactivity. A score of 0 wasconsidered SIRT6^(low) while 1-3 was considered SIRT6^(high). For LIN28Bstaining 0-1 was considered LIN28B^(low) and 2-3 was consideredLIN28B^(high).

RNA In situ Hybridization (RISH) Assay

For the manual (nonautomated) format for ISH, formalin-fixedparaffin-embedded (FFPE) baked tissue sections were subjected toHistoclear deparaffinization (National Diagnostics, Atlanta, Ga.),followed by ethanol dehydration. To unmask the RNA targets, dewaxedsections were incubated in 1× pre-treatment buffer (Affymetrix) (at 90to 95° C. for 10 minutes and digested with 1:100 dilution protease at40° C. for 10 minutes, followed by fixation with 10% neutral bufferedformalin at room temperature for 5 minutes. Unmasked tissue sectionswere subsequently hybridized with 1:30 dilution of the Let7a (AffymetrixVM1-10266) probe for 3 hours at 40° C., followed by a series ofpost-hybridization washes. Signal amplification was achieved by a seriesof sequential hybridizations and washes as described in the View-RNAuser manual (see link below). The specific conditions were as follow:pre-AMP: 25 minutes at 40° C.; AMP: 15 minutes at 40° C.; hybridizationwith labeled probe: 1:1000 dilution for 15 minutes at 40° C.; signaldetection with fast-red substrate: 30 minutes at 40° C. Slides werecounterstained with Gill hematoxylin and mounted using Dako Ultramount(Dako, Carpinteria, Calif.).

For the automated Housekeeping gene (HKG) ISH, the assay was performedby using View-RNA eZL Detection Kit (Affymetrix) on the Bond RXimmunohistochemistry and ISH Staining System with BDZ 6.0 software(Leica Biosystems). FFPE tissue sections on slides were processedautomatically from deparaffinization, through ISH staining tohematoxylin counterstaining. Briefly, 5 mm-thick sections offormalin-fixed tissue were baked for 1 hour at 60° C. and placed on theBond RX for processing. The Bond RX user-selectable settings were asfollows: ViewRNA eZ-1 Detection 1-plex (Red) protocol; ViewRNA DewaxlPreparation protocol; View RNA HIER 10 minutes, ER1 (setting 95);ViewRNA Enzyme 2 (setting 10); ViewRNA Probe Hybridization. With thesesettings, the RNA unmasking conditions for the FFPE tissue consisted ofa 10-minute incubation at 95° C. in Bond Epitope Retrieval Solution 1(Leica Biosystems), followed by 10-minute incubation with Proteinase Kfrom the Bond Enzyme Pretreatment Kit at 1:1000 dilution (LeicaBiosystems). ViewRNA eZ Check-Human TYPE 1 (Affymetrix Cat # DVA1-16742)(a cocktail of GAPDH, PPIB, and ACTB) was diluted 1:40 in ViewRNA ProbeDiluent (Affymetrix). Post run, slides were rinsed with water, air driedfor 30 minutes at room temperature and mounted using Dako Ultramount(Dako, Carpinteria, Calif. ), and visualized and photographed with anOlympus DP72 microscope. Punctate like red color hybridization signalsin the cell nuclei and cytoplasm were defined as positive signal. Slideswere scored semi quantitatively in a blinded manner on a 0-3 scale basedon the intensity of reactivity. If punctate red dots could be visualizedusing the 2× or 4× microscope objective then the section was given ascore of +3, at 10× a score of +2, at 20× or 40× a score of +1 andfinally no signal at 40× was given a score of zero. A score of zero wasconsidered let-7a low and a score of 1-3 was considered let-7a high.

Proliferation Assay

Cells were plated in duplicate on collagen-coated 6-well dishes (1×10⁴per well) in culture medium. Adherent cells were harvested, trypsinizedand counted by trypan-blue exclusion using a Countess Automated CellCounter (Invitrogen) 24, 72, or 120 hours later. Proliferation assayswere performed in duplicate and are represented as mean±s.e.m. betweenthree independent experiments. Alternatively, cells were plated intriplicate in 96-well plates (2000 cells/well for human PDAC cells and500 cells/well on collagen coated plates for murine PDAC cells) inculture medium. MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay wasperformed each day for six days. MTT (1.25 mg/mL final concentration)was added to the culture media and incubated for 3 hours. Formazancrystals were solubilized with 100 μL/well of DMSO and absorbance wasread at 570 nm. To determine the sensitivity of cells todichloroacetate, 500 cells/well were seeded on collagen coated plates inculture medium. The following day dichloroacetate was added to theculture medium and cells were allowed to grow for five days after whichMTT was added to the culture medium as described above. MTTproliferation assays were performed in triplicate and are represented asmean±s.e.m. between three independent experiments unless otherwise notedin the figure legend.

Apoptosis Assay

Cells were washed with PBS and resuspended in 50 μl of 1X Binding Buffer(10 mM HEPES, pH 7.4; 140 mM NaCl; 2.5 mM CaCl2). 2.5 μl of AnnexinV-FITC was added to each sample and incubated 15 minutes in the dark.After this time, 450 μl of 1X Binding Buffer was added and Annexin Vpositive cells analyzed by flow cytometry. Data are shown as mean±stdbetween triplicates and are representative of two independentexperiments.

Caspase 3/7 Activity

Cells were plated at confluency (10,000 cells/well) and allowed toadhere for 24 hours in 96-well plate format. The following day caspase3/7 activity was assessed using a Caspase-Glo® 3/7 Assay (G8090,Promega) per the manufacturer's recommended protocol. Data are shown asmean±std between triplicates.

Cell Cycle Analysis

Briefly, cells were resuspended in 500 μl of PBS and fixed in ethanol byadding drop-wise 1 ml of 95% ethanol. Fixed cells were incubated at 4 Covernight, washed with PBS and resuspended in 500 μl of PBS-0.1%Triton-X-100 supplemented with 1 μg/ml of RNAse A and 20 μg/ml ofpropidium iodide. Samples were incubated at 37° C. for 20 minutesfollowed by 1 hour at 4° C. and DNA content analyzed by flow cytometry.Cell cycle analysis was performed using the ModFit LT software. Data areshown as mean±std between triplicates and are representative of twoindependent experiments.

Chromatin Immunoprecipitation

CMP and qRT-PCR were performed as previously described (Donner et al.,2007). The antibodies used were anti-H3K9Ac (Millipore 07-352),anti-H3K56Ac (ab76307). Quantitative RTPCR for CMP analyses wereperformed in duplicate and are represented as mean±s.e.m. between twoindependent experiments.

Chromatin Immunoprecipitation Sequencing

The steps of CMP followed by sequencing (ChIP-seq) were performed usinga modified version of our previous protocols adapted to the Bravo liquidhandling platform (Agilent) as previously described (Etchegaray et al.,2015). Mouse PDAC cells were crosslinked, fixed cells were lysed and thechromatin was then sheared on Covaris E-220 to a size range between 200and 800 bp. An anti-H3K56Ac (ab76307) antibody was used. The antibodywas incubated a mix of Protein-A and Protein-G Dynabeads (Invitrogen)then incubated overnight. Next, samples were washed eluted, reversecrosslinked, treated with RNaseA (Roche) and Proteinase K (NEB).Illumina library construction reactions were performed as describedpreviously (Etchegaray et al., 2015), using the Bravo liquid handlingplatform (Agilent).

H3K56ac ChIP-Seq Data Analysis

Reads from H3K56ac ChIP-Seq for RP07 SIRT6 wild type (S6WT), SK03 SIRT6knock-out (S6KO), and SK03 SIRT6 knock-out with SIRT6 restored (S6WT)(referred to as samples S1, S2, and S3 respectively) from mousepancreatic cancer cell lines were aligned to mouse genome mm9 using bwa(Li and Durbin, 2009) and duplicate reads were marked with Picard tools(picard.sourceforge.net). Peaks were called using MACS2 (version 2.0.10)with a False Discovery Rate (FDR) set to 0.01 (Zhang et al., 2008). UCSCMus musculus mm9 refGene gene annotation was used to generate a list oftranscription start sites (TSS) for all genes and used to generate a bedfile with TSS +/− 1 kb regions for all genes. The bedtools programintersectBed (bedtools.readthedocs.org/en/latest/index.html) was used toassociate the MACS2 peaks from the three H3K56ac ChIP-Seq samples withthe TSS +/− 1 kb regions and identify genes with peaks. Intersections ofthese gene lists were used to generate the Venn diagrams of FIG. 3A.Genes from the 184 gene subset with TSS +/− 1 kb peaks in S2 SK03 S6KObut not in S1 RP07 S6WT and not it S3 SK03 S6WT were prioritized forfollow-up according to differential binding of H3K56ac ChIP-Seq readsfrom S2 SK03 S6KO relative to S1 RP07 S6WT. The R Bioconductor programDBChIP (Liang and Keles, 2012) was used to analyze the differentialbinding at the TSS locations associated with the 184 S2 but not S1 or S3peaks. DBChIP was run with a window size of 1000, fragment lengths of229 and 219 from the MACS2 estimate of fragment length for S1 and S2respectively, and library sizes of 19319155 and19059445 from the numberof tags after filtering.

Gene Expression Analysis and GSEA

Gene expression data from pancreatic cancer datasets used for the GeneSet Enrichment Analysis (GSEA) in FIGS. 7B-D are accessible from GEO(ncbi.nlm.nih.gov/gds/) (91 pancreatic cancer tumor samples fromPerez-Mancera et al. 2012 (GSE36294) (Perez-Mancera et al., 2012), 36pancreatic cancer tumor samples from Pei et al. 2009 (GSE16515) (Pei etal., 2009), 45 pancreatic cancer tumor samples from Zhang et al. 2012(GSE28735) (Zhang et al., 2012), and 36 pancreatic ductal adenocarcinomatumor samples from Badea et al. 2008 (GSE15471) (Badea et al., 2008)),177 pancreatic adenocarcinoma (PAAD) primary tumor samples from TCGA(cancergenome.nih.gov/), 269 pancreatic cancer ductal adenocarcinoma(PACA-AU v18) tumor samples from Australian pancreatic ICGC (icgc.org)(Waddell et al., 2015), and 43 pancreatic cancer cell line samples fromCCLE (broadinstitute.org/software/cprg/?q=node/11) (Barretina et al.,2012). For the GEO patient sample data sets GSE16515, GSE28735, andGSE15471 and the CCLE data set, raw expression values in the form of CELfiles were collected and then processed using RMA in the R Bioconductorpackage. For the GEO patient sample data set GSE36924 the series matrixfile for the Illumina HumanHT-12 V4.0 expression beadchip data wasdownload. For TCGA data, expression data sets were created by combiningRNASeqV2 Level3 normalized gene result files for individual samples andproducing tables with genes in rows and samples in columns. ICGC datawas read in from the ICGC PACA-AU v18 array fileexp_array.PACA-AU.tsv.gz. Gene Set Enrichment Analysis (Mootha et al.,2003; Subramanian et al., 2005) was used to evaluate the association ofLIN28B expression with known pathways and phenotypes. GSEA was run usingPearson correlation with LIN28B to rank genes and p-values were obtainedfrom 2500 permutations of the LIN28B expression phenotype. GSEA wasperformed using two libraries from version 4.0 of the molecularsignature database (MolSigDB)(broadinstitute.org/gsea/msigdb/index.jsp): the c2 curated gene setsfrom online pathway databases, PubMed publications, knowledge of domainexperts and the c3 motif gene sets.

Statistics

For qRTPCR analysis, proliferation assays, glucose uptake, tumor sphereformation, and tumor size, significance was analyzed using 2-tailedStudent's t test. A p-value of less than 0.05 was consideredstatistically significant. A log-rank test was used to determinesignificance for Kaplan-Meier analyses. Fisher's exact t test wasperformed for comparison of metastatic disease burdens.

Example 1 Loss of SIRT6 Cooperates with Oncogenic Kras to AcceleratePDAC

To determine the tissue expression pattern of SIRT6 in human PDACtumors, tissue microarrays containing 120 pathologist-verified andclinically annotated PDAC samples were generated, including specificcohorts of 40 short-term (<6 months) and 40 long-term (>3 years)survivors. Staining of these samples using a validated antibody forSIRT6 revealed that ˜30-40% of PDAC tumors demonstrated reduced SIRT6expression compared to normal pancreas (FIG. 1A). Although the prognosisfor this disease is already quite poor, patients who underwent surgicalresection of a SIRT6^(low) PDAC tumor had an even worse prognosis inthis retrospective analysis, with a median overall survival of 17.5months compared to 33 months in the SIRT6^(high) tumors (FIG. 1B). Thefunctional role of SIRT6 by knocking down SIRT6 in human pancreaticductal epithelial (HPDE) cells was then evaluated. These studiesrevealed that SIRT6 actively represses both global levels of acetylatedH3K56 and cellular proliferation in pancreatic ductal cells, promptingfurther exploration into the role of SIRT6 in the pathogenesis of PDACin a physiologic context.

To determine whether SIRT6 delays the development of PDAC in agenetically engineered mouse model (GEMM), Sirt6 conditional knockoutmice (Sirt6^(fl/fl)) were crossed with mice harboring apancreas-specific Cre recombinase (p48-Cre), a floxed p53 allele(p53^(fl/+)), and a lox-STOP-lox (LSL) Kras_(G12D) allele to generateLSL-Kras^(G12D); p48-Cre mice with specific loss of one or both Sirt6and p53 alleles in the pancreas. Remarkably, in the context of activatedKras in the pancreas, loss of Sirt6 greatly accelerated the developmentof lethal pancreatic tumors regardless of p53 status (FIGS. 1C-H). Inaddition to developing PDAC and high-grade pancreatic intraepithelialneoplasia (PanIN) at an earlier age, Sirt6-deficient tumors had agreater propensity to metastasize to the lung, compared to their Sirt6wild-type (WT) counterparts (FIGS. 1D-E and 1G-H). Importantly, theseresults demonstrate that SIRT6 suppresses both the formation andmetastasic spread of KRAS^(G12D)-driven PDAC and establish SIRT6 as acritical tumor suppressor in this disease.

Example 2 SIRT6 Suppresses Proliferation of Established PDAC ThoughHistone Deacetylation

To understand how the loss of this bioenergetic sensor influences thebiology of established tumor cells, cell lines were derived fromSirt6^(f/f); Kras^(G12D); p53^(f/+); p48-Cre (SIRT6 KO) and Sirt6^(+/+);Kras^(G12D); p53^(f/+); p48-Cre (SIRT6 WT) murine tumors.

Interestingly, SIRT6 KO PDAC cell lines were highly enriched for tumorsphere forming cells compared to SIRT6 WT cells grown under restrictiveculture conditions, which suggested an enhanced tumorigenic potential(FIG. 2A). In accordance with the role of SIRT6 as a histone deacetylaseand repressor of Myc-mediated transcription (Sebastian et al., 2012),PDAC cells lacking SIRT6 had increased global levels of H3K56Ac as wellas increased chromatin-bound Myc compared to SIRT6 WT cell lines,although total levels of Myc in the whole cell lysate were similarbetween the two genotypes (FIG. 2B). The direct role of SIRT6 histonedeacetylase activity in regulating these phenotypes was confirmed by thefact that WT but not catalytically inactive SIRT6 (S6HY) reduced globallevels of H3K56Ac, chromatin-bound Myc, cell proliferation and tumorsphere formation (FIGS. 2B-D). Finally, restoration of SIRT6 expressionin SIRT6 KO PDAC cells also slowed tumor growth in vivo (FIG. 2E). Tovalidate these findings in human PDAC, SIRT6 levels were analyzed in apanel of patient-derived PDAC lines. Consistent with the analysis ofhuman PDAC tissues, SIRT6 protein levels were reduced in 6 out of 13(46%) of the human PDAC cell lines surveyed when compared to HPNE cells(FIG. 2F). Restoration of SIRT6 expression in human PDAC cell lines thatexhibit reduced levels of SIRT6 (SIRT6^(low)) suppressed H3K9Ac andH3K56Ac levels, cell cycle progression and cellular proliferation, whileinducing apoptosis and robustly inhibiting tumor sphere formation in amanner that required SIRT6 deacetylase activity (FIGS. 2G-J). Thus, theloss of this NAD⁺-dependent histone deacetylase leads tohyperacetylation of chromatin and increased cellular proliferation inboth normal pancreatic ductal cells and PDAC.

SIRT6 is a central regulator of glycolytic metabolism (Sebastian et al.,2012; Zhong et al., 2010). Consistent with this finding, knockdown ofSIRT6 in HPDE cells resulted in increased expression of HIF1α targetgenes involved in glycolytic metabolism, such as pyruvate dehydrogenase1 (PDK1), lactate dehydrogenase a (LDHA), and the glucose transporter(GLUT1) (FIGS. 2K and 2L). These gene expression changes correspondedwith an increase in uptake of the fluorescently labeled glucose analog2-(N-7-nitrobenz-2-oxa-1,3,diazol-4-yl)amino)-2-deoxyglucose (2-NBDG)(FIG. 2M). Conversely, when SIRT6 levels were restored in SIRT6^(low)PDAC cell lines, glycolytic gene expression and glucose uptake were allrepressed (FIGS. 2N-P). Likewise, SIRT6 KO PDAC cells demonstratedrelatively high expression of Pdk1, Ldha and Pfkm as well as 2-NBDGuptake compared to SIRT6 WT cells, and expression of SIRT6 reducedglycolytic gene expression. However, despite these increased levels ofglucose uptake and glycolytic gene expression, knocking down Pdk1 orLdha, both central regulators of glycolytic metabolism, had equivalenteffects on SIRT6 WT and KO PDAC cells. In addition, pharmacologicinhibition of PDK1 with the small-molecule PDK1 inhibitor,dichloroacetate (DCA), inhibited growth of both SIRT6 WT and KO PDACcell lines with similar potency. These results suggested that lack ofSIRT6 does not render PDACs more sensitive to glycolysis inhibition. Tofully evaluate the role of glycolysis in the accelerated formation ofSIRT6 KO PDAC, SIRT6 KO and WT mice were treated with DCA in theirdrinking water from 4 weeks of age and monitored for the development oflethal PDAC tumors. Consistent with our in vitro results, DCA treatmentminimally delayed the onset of SIRT6 KO PDAC. Overall, these resultsindicate that enhanced glycolysis plays a modest role in the increasedaggressiveness of these SIRT6-deficient tumors, in contrast to what waspreviously observed in colon cancer (Sebastian et al., 2012).

Example 3 SIRT6 Suppresses Expression of the Oncofetal Protein Lin28b inHuman and Murine PDAC

The resistance of SIRT6 KO PDAC cells to Pdk1 knockdown and the failureto reverse the SIRT6 KO phenotype with DCA treatment prompted us toinvestigate alternative pathways regulated by SIRT6 that could limit thegrowth of PDAC cells. Since expression of WT but not catalyticallyinactive SIRT6 slowed the growth of both human and murine PDAC cells, wehypothesized that these pathways would be regulated by the histonedeacetylase activity of SIRT6. We therefore sought to identify novelgenes regulated by SIRT6 histone deacetylase activity by performingchromatin immunoprecipitation (ChIP) of H3K56Ac marks (the mainchromatin substrate of SIRT6) followed by next generation sequencing(ChIP-seq) on SIRT6 WT and SIRT6 KO murine PDAC cells, as well as SIRT6KO cells engineered to express WT SIRT6 (SIRT6 KO+SIRT6 WT). In SIRT6 KOcells, a total of 12,049 genes were identified that are decorated withH3K56Ac within 1 kilobase (kb) of their transcription start site. Toidentify genes that were dynamically regulated by SIRT6, genes wereisolated that were only marked in SIRT6 KO cells but not SIRT6 WT cells,and which lost this mark upon re-expression of SIRT6 (FIG. 3A). Theremaining 184 gene promoters were then ranked based on fold change ofH3K56Ac in SIRT6 KO compared to SIRT6 WT cells. Intriguingly, theRNA-binding protein Lin28b was the top candidate in this list (FIG. 3B).

Although highly expressed in embryonic tissues, Lin28b is fully silencedduring differentiation and in healthy adult cells (Moss and Tang, 2003;Rybak et al., 2008; Yang and Moss, 2003), but may be aberrantlyreactivated in a variety of human cancers (Iliopoulos et al., 2009;Thornton and Gregory, 2012; Viswanathan et al., 2009). While Lin28b hasbeen correlated with advanced disease and poor prognosis (King et al.,2011; Lu et al., 2009; Viswanathan et al., 2009), its functional roleand mechanism of reactivation in human cancer remain poorly understood.Furthermore, neither Lin28b expression, its regulation nor itsfunctional role in PDAC have previously been explored. Although the Myctranscription factor can bind to consensus sequences within the Lin28bpromoter (Chang et al., 2009), overexpression of Myc does not seemsufficient to drive its expression, suggesting that additional cofactorsor epigenetic modifications are required (Viswanathan and Daley, 2010).The high levels of H3K56Ac over the Lin28b gene promoter in SIRT6 KOPDAC cells prompted us to explore whether loss of the epigeneticmodifier SIRT6 may be one such mechanism of reactivation and whether theexpression of Lin28b may drive the growth of a specific subset of PDAC.

Strikingly, all SIRT6 KO PDAC mouse lines analyzed exhibited far higherLin28b expression than SIRT6 WT PDAC lines, both at the RNA and proteinlevel (FIGS. 3C and 3D). Similar differences were seen in vivo, as PDACtumors from SIRT6 KO mice were also universally positive for LIN28B,while SIRT6 WT PDAC tumors demonstrated only background levels ofstaining for LIN28B by immunohistochemistry. Remarkably, expression ofSIRT6 and LIN28B were also inversely correlated in human PDAC cell linesby quantitative real-time PCR (qRT-PCR) (FIGS. 3E and 3F). To define thephysiological significance of these observations, expression of LIN28Bwas analyzed directly in our panel of 120 human PDAC patient samples.Consistently, tumors with low or undetectable levels of SIRT6 exhibitedrobust staining for LIN28B (FIGS. 3G). Lastly, ectopic expression of WT,but not catalytically inactive SIRT6, suppressed expression of LIN28B inPanc3.27 cells (FIGS. 3H and 3I) and in 2 independent murine SIRT6 KOPDAC lines (FIG. 3J) confirming that loss of SIRT6 leads to thereactivation of Lin28b in both human and murine PDAC. Interestingly,SIRT6 may also regulate Lin28b expression in non-epithelial tissues asrestoration of SIRT6 reversed the elevated levels of Lin28b expressionobserved in an immortalized murine embryonic fibroblast (MEF) cell line(Mostoslaysky et al., 2006).

Example 4 SIRT6 Co-Represses Myc-Driven Transcription of Lin28b ThroughHistone Deacetylation

SIRT6 represses Myc-dependent transcription by deacetylating histonemarks, resulting in an inaccessible chromatin structure (Sebastian etal., 2012). Therefore, chromatin immunoprecipitation (ChIP) assays wereused to interrogate whether SIRT6 may co-repress Myc at the Lin28blocus. Interestingly, SIRT6 KO cells had significantly increased levelsof H3K56Ac and H3K9Ac compared to SIRT6 WT cells at two known Mycbinding sites within the Lin28b promoter, suggesting an open andpermissive chromatin conformation (FIGS. 4B-C). Direct binding of SIRT6to these Myc binding sites within the Lin28b promoter was confirmed inSIRT6 KO MEFs transfected with either WT SIRT6 or S6HY, whereas only WTSIRT6 could remove H3K56Ac and H3K9Ac marks in this region. Furthermore,we found that this was a dynamic and constitutive process in human PDACcells with high levels of SIRT6 (SIRT6^(high)), such as COLO357 cells,where acute loss of SIRT6 by shRNA-mediated knockdown resulted inincreased H3K9 and H3K56 acetylation in a homologous region of the humanLIN28B promoter. The critical role of Myc in driving Lin28b expressionin PDAC was confirmed by knocking down Myc in three independent SIRT6 KOmurine and SIRT6^(low) human PDAC cell lines, which resulted in aconsistent and proportional reduction in Lin28b expression (FIGS. 4D and4E). Consistent with their antagonistic relationship, Myc knockdownphenocopied restoration of SIRT6 expression in both SIRT6 KO murine andSIRT6^(low) human PDAC cells, where reduced cellular proliferation ratesand tumor sphere formation were observed (FIGS. 4F-J). Taken together,these data strongly support a mechanism by which SIRT6 activelyco-represses Myc-dependent transcription in both human and murine PDACspecifically at the Lin28b locus, through deacetylation of H3K56 andH3K9 chromatin marks.

Example 5 SIRT6^(low) PDAC Cells are Addicted to Lin28b

The functional role of Lin28b in SIRT6 KO murine PDAC cells andSIRT6^(low) human PDAC cells were examined by knocking down Lin28b withboth shRNA and siRNA, which resulted in potent suppression of cellproliferation and tumor sphere formation in two independent murine SIRT6KO cell lines, while two independent SIRT6 WT PDAC lines were completelyinsensitive to the same treatment. More importantly, both shRNA andsiRNA against LIN28B also markedly reduced the proliferation, tumorsphere forming ability and in vivo xenograft growth of several humanSIRT6^(low) PDAC lines without any discernable effect on the growth ofhuman PDAC lines with normal levels of SIRT6 (SIRT6^(high)) (FIGS.5D-H). As with restoration of SIRT6 expression, knockdown of Lin28b ledto both G1 cell cycle arrest and induction of apoptosis in twoindependent SIRT6^(low) lines. Thus, LIN28B is both upregulated andcritically required for the growth and survival of this subset of PDACcancers, as defined by loss of SIRT6 expression.

Example 6 Let-7 Suppresses Igf2bps and Hmga2 Expression and PDAC CellGrowth

The most well-characterized function of Lin28b is to inhibit thebiogenesis of a family of 12 tumor suppressor microRNAs (miRNAs),collectively referred to as let-7 (Heo et al., 2008; Newman et al.,2008; Pasquinelli et al., 2000; Rybak et al., 2008; Viswanathan et al.,2008). Mature let-7 miRNAs are found in a reciprocal pattern withLin28b, suppressed in embryonic tissues and highly expressed in normaladult cells (Bussing et al., 2008; Moss and Tang, 2003; Rybak et al.,2008; Yang and Moss, 2003), where it can promote the degradation of anumber of targets involved in carcinogenesis (Johnson et al., 2005; Mayret al., 2007; Sampson et al., 2007), including Insulin Growth Factor 2Binding Proteins (IGF2BPs) and High Mobility Group AT-Hook 2 (HMGA2)(Boyerinas et al., 2008; Mayr et al., 2007; Nguyen et al., 2014; Park etal., 2007; Polesskaya et al., 2007). To determine whether Lin28b maydrive the growth and survival of PDAC cells through the inhibition oflet-7, the levels of let-7 miRNA family members were measured followingLin28b knockdown in our human SIRT6^(low) PDAC cells. Indeed, theexpression of almost all let-7 family members increased following Lin28bknockdown. To assess the functional significance of this let-7reactivation, exogenous mimetics of the let-7c and let-7d family memberswere transfected into human PDAC cells, and they specifically inhibitedthe growth of the SIRT6^(low) cell lines BxPc3 and Panc-1 without anysignificant effect on the growth of the SIRT6^(high) cell line COLO357.We also obtained a miRNA which mimics let-7g, but has been mutated sothat it is unable to be bound and degraded by Lin28b (Piskounova et al.,2008). Ectopic expression of this let-7g mimetic (7S21L) potentlyinhibited both proliferation and tumor sphere forming ability ofSIRT6^(low) PDAC cell lines. Importantly, growth inhibition followingectopic expression of let-7 mimetics was also accompanied by a reductionin the expression of let-7 target genes, IGF2BP1, IGF2BP3, and HMGA2 aswell as LIN28B, which is also directly inhibited by let-7 as a part of afeedback loop (Rybak et al., 2008). Thus, multiple let-7 family memberspotently and selectively inhibit the growth of SIRT6^(low) PDAC cells,potentially through the suppression of let-7 target genes.

Example 7 SIRT6^(low) PDAC are Dependent on let-7 Target Genes forGrowth

Interestingly, high expression of Igf2bp1 and Igf2bp3, which are bothdirectly inhibited by let-7, is correlated with increased aggressivenessand metastasis in pancreatic tumors (Thakur et al., 2008; Yantiss etal., 2005), and in support of a causal role in transformation,transgenic overexpression of mouse Igf2bp3 (IMP3/KOC) leads to increasedcell proliferation and metaplasia of pancreatic acinar cells (Wagner etal., 2003). Both Igf2bp1 and Igf2bp3 were highly upregulated in ourSIRT6 KO cells relative to SIRT6 WT cells and in our human SIRT6^(low)PDAC cells (FIG. 6A). In addition, HMGA2 is another let-7 target genethat is associated with advanced tumor grade and lymph node metastasisin PDAC (Piscuoglio et al., 2012) and, strikingly, was universallyexpressed in SIRT6^(low) lines, but almost completely absent in allSIRT6^(high) lines analyzed (FIG. 6A). Restoration of WT but notcatalytically inactive SIRT6 in SIRT6 KO murine and SIRT6^(low) humanPDAC cells reduced expression of both Lin28b and let-7 target genes,confirming the direct role of SIRT6 in regulating this pathway. Whilethese findings are consistent with a model whereby SIRT6 acts upstreamof the Lin28b/let-7 axis to suppress Lin28b expression, enhance let-7levels and inhibit expression of let-7 target genes, the functional roleof each of these let-7 target genes in driving the growth of PDAC cellshas not yet been clearly established. Therefore, either HMGA2 or IGF2BP3was knocked down in a panel of human PDAC cell lines. Remarkably,although the Lin28b/let-7 pathway has many known targets, knock-down ofeither HMGA2, IGF2BP1, or IGF2BP3 was sufficient to inhibit bothproliferation and tumor sphere formation in SIRT6^(low) PDAC cellswithout any discernable effect on SIRT6^(high) PDAC cells (FIGS. 6B-H).Further, knock-down of Igf2bp3 with siRNA specifically slowed growth ofSIRT6 KO cells but had no effect on SIRT6 WT murine PDAC cells. Thus,multiple let-7 target genes may cooperate to drive the growth ofSIRT6^(low) PDAC.

Example 8 Increased Expression of LIN28B and let-7 Target GenesCorrelates with Poor Survival in PDAC

These observations prompted us to investigate the relevance of thispathway to the human disease. As shown previously, loss of SIRT6expression in human PDAC tumors defined a subset of patients with aworse prognosis (FIG. 1B). Strikingly, elevated expression of LIN28Balso correlated with poor prognosis in our same cohort of 120 patientsamples (FIG. 7A). Moreover, gene set enrichment analysis (GSEA)comparing PDAC tumors (Badea et al., 2008; Biankin et al., 2012; Pei etal., 2009; Perez-Mancera et al., 2012; Zhang et al., 2012) and celllines (Barretina et al., 2012) with high versus low expression of LIN28Brevealed that LIN28B^(high) tumors were strongly enriched for theexpression of Myc targets, as well as for let-7 targets, curated inthree independent gene sets (FIG. 7B). This finding was furthervalidated in the CCLE dataset (FIG. 7C). More specifically, theoncofetal targets of let-7, which includes the IGF2BPs and HMGA2, wereupregulated in LIN28B^(high) tumors in three independent datasets (FIG.7D). Accordingly, loss of let-7 expression, as measured by in-situhybridization (ISH) for let-7a, also corresponded to a shorter overallsurvival. Finally, expression of these oncofetal targets IGF2BP3 andHMGA2 correlated both with each other and a worse prognosis in thecancer genome atlas (TCGA) dataset (FIGS. 7E and 7F). Taken together,our findings are consistent with a model whereby loss of SIRT6 in PDACallows for aberrant hyperacetylation of the Lin28b promoter, enhancingMyc-driven transcription of Lin28b, which then inhibits the let-7 familyof miRNA. This allows for the reactivation of let-7 target genes such asHMGA2 and IGF2BPs, which serve to drive the growth and survival of ahighly aggressive form of pancreatic cancer (FIG. 7G).

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Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1.-5. ( canceled)
 6. A method of treating PDAC in a subject, the methodcomprising administering to the subject a therapeutically effectiveamount of a Lin28b inhibitor, wherein the Lin28b inhibitor comprises aninhibitory nucleic acid effective to specifically reduce expression ofLin28b, thereby treating PDAC in the subject.
 7. The method of claim 6,wherein the inhibitory nucleic acid is a small interfering RNA molecule,antisense nucleic acid, LNA molecule, PNA molecule, or ribozyme.
 8. Themethod of claim 6, wherein the inhibitory nucleic acid comprises asequence of 5′-GCCTTGAGTCAATACGGGTAA-3′ (SEQ ID NO:3).
 9. The method ofclaim 6, wherein the subject is a mammal.
 10. The method of claim 6,wherein the subject is a human.
 11. A method of diagnosing andoptionally treating PDAC in a subject, the method comprising: providinga sample comprising pancreatic cells from the subject; performing anassay to determine a level of Lin28b expression in the sample; comparingthe level of Lin28b expression in the sample to a reference level ofLin28b expression; identifying a subject who has a level of Lin28bexpression in the sample above the reference level as having PDAC orhaving an increased risk of developing PDAC; and optionallyadministering a treatment for PDAC to the identified subject who has alevel of Lin28b expression in the sample that is above the referencelevel, wherein the treatment comprises an inhibitory nucleic acideffective to specifically reduce expression of Lin28b.
 12. The method ofclaim 11, wherein the inhibitory nucleic acid is a small interfering RNAmolecule, antisense nucleic acid, LNA molecule, PNA molecule, orribozyme.
 13. The method of claim 11, wherein the inhibitory nucleicacid comprises a sequence of 5′-GCCTTGAGTCAATACGGGTAA-3′ (SEQ ID NO:3).14. The method of claim 11, wherein the level of Lin28b expression inthe sample is determined by quantitative PCR, flow cytometry, orquantitative immunoassay.
 15. The method of claim 11, wherein thesubject is a mammal.
 16. The method of claim 11, wherein the subject isa human.
 17. A method of diagnosing and optionally treating PDAC in asubject, the method comprising: providing a sample comprising pancreaticcells from the subject; performing an assay to determine a level ofSIRT6 expression in the sample; comparing the level of SIRT6 expressionin the sample to a reference level of SIRT6 expression; identifying asubject who has a level of SIRT6 expression in the sample below thereference level as having PDAC or having an increased risk of developingPDAC; and optionally administering a treatment for PDAC to theidentified subject who has a level of SIRT6 expression in the samplethat is below the reference level, wherein the treatment comprises aninhibitory nucleic acid effective to specifically reduce expression ofLin28b.
 18. The method of claim 17, wherein the inhibitory nucleic acidis a small interfering RNA molecule, antisense nucleic acid, LNAmolecule, PNA molecule, or ribozyme.
 19. The method of claim 17, whereinthe inhibitory nucleic acid comprises a sequence of5′-GCCTTGAGTCAATACGGGTAA-3′ (SEQ ID NO:3).
 20. The method of claim 17,wherein the level of SIRT6 expression in the sample is determined byquantitative PCR, flow cytometry, or quantitative immunoassay.
 21. Themethod of claim 17, wherein the subject is a mammal.
 22. The method ofclaim 17, wherein the subject is a human.